PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as "compliant" or "noncompliant," according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 l showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (>3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 l.

Based on our observations, anidulafungin has effects that are different from the ones produced by micafungin and caspofungin. Whether this different response to high concentrations of echinocandins is based on genetic or phenotypic differences between C. albicans and C. dubliniensis has to be determined in future experiments.

Production of E,E-farnesol (FOH) and biofilm formation were studied under various conditions in 56 strains of eight Candida spp. FOH production differed significantly not only between Candida spp. but within Candida albicans strains as well. FOH concentrations and biofilm formation were the highest for C albicans.Candida albicans is a major human fungal pathogen, causing both superficial and invasive tissue infections. The ability of C. albicans to form biofilms on medical devices has a profound impact on its capacity to cause human disease. C. albicans and other members of the genus Candida are able to grow in different forms as budding yeast, pseudohyphae, and true hyphae, which is called dimorphism (1,8). This transition from yeast to hyphal growth can be induced by various conditions (2, 25). Progression to a mature biofilm is dependent on cell adhesion, extracellular matrix production, and the yeast-to-hypha transition in C. albicans (2, 3). However, biofilm development in non-C. albicans Candida spp. (NCAC) is not well understood.Suppression of biofilm formation in C. albicans may be achieved by quorum-sensing molecules (5). E,E-Farnesol (FOH) has been reported to inhibit the induction of hyphal growth and biofilm formation in C. albicans (10,11). In this study, FOH secretion by C. albicans and eight NCAC was examined under various culture conditions. In addition, the development of biofilms was studied. Finally, the correlation between FOH secretion and biofilm formation was analyzed for all of the isolates studied.We studied 56 strains of eight Candida species (Table 1). All isolates were cultivated in RPMI 1640 medium with or without the addition of 10% fetal calf serum (FCS) at 37°C for 24 h under continuous rotation at 125 rpm. Two milliliters of sterile filtered (0.45 m) culture supernatant was extracted with 5 ml n-hexane-ethanol (90:10, vol/vol) and derivatized with 9-anthroylnitrile as previously described (13). Quantification was done with n-butanol as an internal standard (50 ng added to each sample). Reverse-phase high-performance liquid chromatography was done with a YMC Hydrosphere C 18 column (5 m, 150 by 2.1 mm [inside diameter]). A linear gradient of acetonitrile-water (85% to 100% over 20 min) was used as the mobile phase. Standard concentrations ranged from 0.004 M to 40 M FOH. Detection, determination of recovery, and calculations were performed as previously described (4, 13). The development of biofilms by all of the Candida spp. was studied according to Krom et al. (6), with minor differences such as using 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt instead of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide. In addition to visual reading at 450 nm, optical density was measured at 405 nm for data validation (15). All tests were done twice and analyzed with the two-tailed paired Student t test comparing 0 and 10% FCS cultivations (significance was set at a P value of Յ0.05).The quantification and...

Diseases caused by Candida species are an increasing problem. Candida species are associated with high overall mortality, due to a variety of virulence factors such as the yeast-to-hyphal switch and proteolytic enzymes. The phenomenon of microbial communication known as quorum sensing also seems to play an important role. The main characteristics of the quorum-sensing molecule E ,E -farnesol are well known for C. albicans. The present study focused on two questions. One of them concerned the secretion of E ,E -farnesol by C. albicans and involved a close examination of the effect of the medium (serum) and the origin of the isolates used. The second one dealt with the activity of E ,E -farnesol in non-C. albicans species, such as C. tropicalis and C. parapsilosis, e.g. its impact on biofilm formation and growth. Under serum conditions, C. albicans produced up to 58% more E ,E -farnesol at 37• C than at 30• C. The growth of all isolates was reduced and delayed by the administration of E ,Efarnesol. Of all Candida species, C. tropicalis isolates were most strongly affected by the addition of E ,E -farnesol. Biofilm formation on polystyrene was affected by E ,Efarnesol treatment in all non-C. albicans species and C. albicans. E ,E -farnesol exerts its main effect by altering the metabolic activity and growth inhibition of treated Candida species. The results obtained indicate that the presence of E ,E -farnesol in the environment not only regulates the morphology of the Candida species but also affects its fitness. In this regard, the secretion of E ,E -farnesol might provide an advantage for members of the microbial community.

Resveratrol is a natural stilbene synthesised by plants. This compound has been shown to inhibit the growth of Candida albicans TIMM 1768 efficiently. Till date, no information is available for other Candida species. The evaluation of the antimicrobial activity of resveratrol was analysed by the inhibition of the growth and metabolism assays. Our data indicate that resveratrol is not effective against Candida albicans and non-C. albicans species (C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) in vitro. The potential candidacidal activity could not be confirmed.