On the basis of our results, it appears that the disc diffusion test is a useful method for testing the activity of voriconazole against Aspergillus spp.
The disk diffusion method was evaluated for determining posaconazole susceptibility against 78 strains of molds using two culture media in comparison with the CLSI (Clinical Laboratory Standards Institute) broth microdilution method (M38-A). A significant correlation between disk diffusion and microdilution methods was observed with both culture media.Invasive infections due to Aspergillus spp. and other filamentous fungi (molds) have emerged as prominent causes of morbidity and mortality worldwide in immunocompromised hosts (2,(6)(7)(8)14). Posaconazole is a water-insoluble investigational triazole with in vitro and in vivo activity against yeasts and molds (1, 3, 11, 15). The Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) has developed a reference method for microdilution antifungal susceptibility testing of filamentous fungi (CLSI/NCCLS M38-A) (9). Agar-based methods such as Etest and disk diffusion can be good alternatives because they are simpler and faster than broth-based methods. Some investigators have explored the use of Etest susceptibility testing for posaconazole against filamentous fungi (4, 12), but data on posaconazole testing against molds by the disk diffusion method are lacking. In this study, we investigated the applicability of a disk diffusion method for testing the susceptibility of posaconazole against molds in order to compare the results that were obtained with this assay with those obtained by the broth microdilution method and to evaluate the influence of culture media.A total of 78 clinical isolates were tested: 57 Aspergillus spp. (20 Aspergillus terreus, 16 Aspergillus fumigatus, 14 Aspergillus flavus, 4 Aspergillus niger, and 3 Aspergillus glaucus) and 21 other filamentous fungi (7 Rhizopus spp., 3 Mucor spp., 5 Scedosporium apiospermum, 3 Scedosporium prolificans, and 3 Fusarium spp.). These isolates were recovered from clinical specimens received in Valme University Hospital in Seville (Spain) and the La Fé University Hospital in Valence (Spain). The identification of each strain was performed using conventional mycological techniques. The mold isolates were maintained in sterile water and were subcultured on antimicrobialagent-free potato dextrose agar (Difco) to ensure viability and purity. Stock inoculum suspensions were prepared from 7-dayold cultures grown on potato dextrose agar by following CLSI guidelines (document M38-A) (9). Stock suspensions were adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.11 (80 to 82% transmittance) and contained conidia or sporangiospores and hyphal fragments. The diluted (2ϫ) inoculum sizes ranged from 0.9 ϫ 10 4 to 4.7 ϫ 10 4 CFU/ml, as demonstrated by a quantitative colony count on Sabouraud dextrose agar. The same inoculum was used for broth and agar methods. The broth microdilution test was done in accordance with CLSI guidelines for conidium filamentous fungi (CLSI document M38-A) (9). Posaconazole was obtained from the Schering-Plough R...
We compared the Etest with a broth microdilution method, performed according to a modified National Committee for Clinical Laboratory Standards guideline (M38-A), for determining the in vitro susceptibility of 77 isolates of Aspergillus spp. (26 A. fumigatus, 21 A. flavus, 10 A. terreus, 9 A. niger, 5 A. nidulellus, 4 A. glaucus, and 2 A. flavipes isolates). Overall, there was 92.2% agreement between both methods when Etest MICs were read at 24 h and 83.1% agreement when both methods were read at 48 h. When Etest MICs were read at 24 h, the agreement was >90% for all species tested except for A. fumigatus (84.6%). When Etest MICs were read at 48 h, the agreement ranged from 50 to 100%. The poorest agreement was seen with A. glaucus (50%) and A. fumigatus (65%). Where a discrepancy was observed between Etest and the reference method, the Etest MIC was generally higher. The Etest appears to be a suitable alternative procedure for testing the susceptibility of Aspergillus spp. to voriconazole.
SummaryThe definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and nonpathogenic bacteria ⁄ fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10 1 -10 5 conidia ml )1 ), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5-20 conidia using conidial suspensions. The linear range was from 60 to 6 · 10 7 fg. The Tm ranged from 67.34 to 70.7°C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.
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