P1973 Comparison of RPMI and AM3 medium for in vitro susceptibility testing of caspofungin against Candida spp. by EUCAST broth microdilution method

New antifungals and antifungal resistance Antifungal agent

Amphotericin B Flucytosine Fluconazole Itraconazole Voriconazole Ravuconazole Posaconazole Caspofungin

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C. neoformans var. gattii C. neoformans var. (n = 20) neoformans (n = 333) GM

MIC90

MIC50

GM

MIC90

MIC50

0.09 1.07 14.93 0.45 0.47 0.53 0.26 16

0.125 2 16 1 1 2 0.5 16

0.125 1 16 0.5 0.5 0.5 0.25 16

0.24 4.38 7.44 0.25 0.12 0.15 0.16 18.02

1 16 16 1 0.5 1 0.5 32

0.25 4 8 0.25 0.125 0.125 0.25 16

P1971 Induction of resistance and cross-resistance of fluconazole, voriconazole, caspofungin and cationic antimicrobials against Candida species S. Tobudic, C. Kratzer, W. Graninger, A. Georgopoulos (Vienna, AT) Objectives: The purpose of this study was to compare the in vitro selection of resistance and cross-resistance of fluconazole, voriconazole, caspofungin and the cationic antimicrobials chlorhexidine digluconate and Akacid plusR against Candida species. Methods: For the in vitro selection of resistance Candida albicans ATCC 10231, C. albicans ATCC 90029, Candida glabrata ATCC 90030, Candida krusei ATCC 6258 and Candida tropicalis ATCC 750 were subcultured in liquid RPMI medium containing selected concentrations of active substances during 30 passages. Minimal inhibitory concentrations (MICs) of antimicrobials were performed after each passage using broth microdilution method according to the CLSI guidelines (M27/A2). Results: Subcultivation of C. tropicalis in RPMI containing 32 mg/L fluconazole resulted in a 256–fold increase in the MICs of fluconazole and voriconazole after less than 10 passages. Following exposure to 4 mg/L voriconazole, comparable MIC increases of voriconazole and lower increases of fluconazole were found (128 and 16 times of the MIC) in C. tropicalis. Preexposure to subinhibitory concentrations of caspofungin induced a 64-fold MIC-increase of caspofungin in C. tropicalis following 10 passages, but no cross-resistance to azoles or cationic antimicrobials was observed. Subcultivation of both strains of C. albicans in 2 mg/L voriconazole induced a high MIC increase (>256×) after 10 passages, whereas no cross-resistance to other antimicrobials could be induced. MICs of active substances against C. glabrata and C. krusei remained constant during 30 passages. Development of resistance or cross-resistance to Akacid plusR or chlorhexidine was not observed in any of the tested isolates. Conclusion: Repeated exposure to subinhibitory concentrations of cationic antimicrobials had no effects on selection of resistance or cross-resistance in Candida spp., whereas resistance and cross-resistance to azoles and resistance to caspofungin could be easily induced in C. tropicalis. In vitro resistance of voriconazole was also developed in both strains of C. albicans. P1972 Broth microdilution, Etest and disk diffusion methods for Trichosporon asahii susceptibility testing against established and new antifungal agents M. Kanellopoulou, D. Adamou, M. Martsoukou, N. Skarmoutsou, E. Alexopoulos, A. Milioni, G. Arsenis, A. Velegraki, E. Papafrangas (Athens, Patras, GR) The genus Trichosporon is a basidiomycetous yeast phylogenetically close to Cryptococcus. T. asahii is an emerging cause of disseminated trichosporonosis. Isolation of T. asahii from patient indwelling devices and biological secretions, without related dissemination, is hitherto of ambiguous clinical significance.

Objectives: (a) To monitor over 3 years (2002–2005) T. asahii susceptibility to antifungals isolated from urinary tract and bronchial secretions of patients without trichosporonosis; (b) to compare broth microdilution (BMD), Etest and disk diffusion (DD) methods for determining susceptibility to established antifungals and to posaconazole. Methods: Seventeen urine specimen isolates from patients with indwelling catheter (n = 15) and from respiratory secretions with endotracheal catheter (n = 2) were tested against posaconazole (POS; Schering Plough Research Institute, New Jersey, USA), amphotericin (AB; Sigma, St. Louis, MO, USA), flucytosine (FC; Sigma), itraconazole (IT; Janssen, Beerse, Belgium) fluconazole and voriconazole (FLU and VOR; Pfizer, Sandwich, Kent, UK). Strains were tested by BMD (CLSI, M-27A2) and Etest (AB Biodisk, Solna, Sweden). FLU and POS were also tested by DD (CLSI, M-44A, 2004) using commercially prepared disks (OXOID, Basingstoke, UK). Incubations were performed at 35ºC and read at 24, 48 and 72 hours. The QC isolates used were: Candida krusei ATCC6258 and C. parapsilosis ATCC22019. Results: MIC ranges (mg/mL) were AB: 0.5−16, FC: 2−64, IT: 0.25−2, FLU: 2−32, VO: 0.215−1 and POS: 0.125−0.5. No intrazonal growth was observed in the DD method. Inhibition zone diameters ranged from 5 to 35 mm (FLU) and 10 to 20 mm (POS). Interclass correlation coefficients (ICCs) and 95% confidence intervals for comparing the methods were calculated using log2 transformed data. ICC for BMD versus Etest ranged from 0.98 to 0.99 (P < 10−4 ) for all drugs. Pearson’s correlation coefficient used as a measure of linear associations for FLU and POS BMD versus Etest, versus disk diffusion was 0.944 (P < 0.01). Conclusion: High AB, FC, IT and FLU MICs warrant clinical alertness for breakthrough Trichosporon infections when used for prophylaxis. Pending clinical studies, the low VOR and POS MICs suggest that they may be a better alternative for prophylaxis in patients at risk. Disk diffusion is an option for rapidly testing T. asahii isolates, provided it is further evaluated. Standardised susceptibility testing can aid the selection of the most relevant antifungal therapy for the management of trichosporonosis. P1973 Comparison of RPMI and AM3 medium for in vitro susceptibility testing of caspofungin against Candida spp. by EUCAST broth microdilution method E. Dannaoui, O. Lortholary, D. Raoux, D. Hoinard, F. Dromer (Paris, FR) Objectives: To compare two media, RPMI and AM3, for antifungal susceptibility testing of caspofungin against Candida spp. Methods: A total of 863 Candida spp. isolates belonging to 25 different species were evaluated. All isolates were tested for caspofungin susceptibility in RPMI and AM3. All isolates were identified according to standard procedures. Susceptibility testing was performed according to the EUCAST broth microdilution reference method. Both media were supplemented with 2% glucose with a final inoculum size of 1×105 CFU/mL. Microplates were read spectrophotometrically after 24 h of incubation at 35ºC. MICs were defined as the lowest concentration resulting in 50% or more reduction of growth compared to the drugfree growth control. Quality control strains were included in each set of MIC determination. Results: Overall, lower caspofungin MICs were obtained in AM3 (CMI50 = 0.03 mg/mL) compared to RPMI (CMI50 = 0.5 mg/mL) and this was true for all tested species. There was a broader range (i.e. number of log2 dilutions between the highest and the lowest MIC) of MICs in AM3 than in RPMI for C. albicans (11, and 6 dilutions for AM3 and RPMI, respectively), C. glabrata (11, and 6), C. parapsilosis (6, and 4), and C. tropicalis (7, and 4). Moreover, more isolates exhibited MIC > MIC90 when AM3 was used compared to RPMI for C. albicans, C. glabrata, and C. parapsilosis, suggesting that AM3 is more suitable than RPMI for detection of resistance to caspofungin. Conclusions: For caspofungin susceptibility testing with the EUCAST reference method, the use of AM3 instead of RPMI yielded lower MICs as well as a broader range of MICs. Our results suggest that AM3 could allowed better detection of caspofungin resistant isolates. Further studies are needed to confirm these preliminary results.

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17th ECCMID / 25th ICC, Posters

Table 1. Detection of Candida spp. isolates with high caspofungin MICs in RPMI and AM3 medium Species (n)

C. C. C. C. C.

Caspofungin

albicans (404) glabrata (157) parapsilosis (109) tropicalis (62) krusei (21)

Z.U. Khan, S. Ahmad, A. Theyyathel (Kuwait, KW)

MIC90 (mg/mL)

No. of isolates with MIC > MIC90

RPMI AM3

RPMI

AM3

0.5 1 2 1 2

14 1 5 3 1

23 11 6 2 1

0.03 0.06 0.5 0.06 0.125

P1974 In vitro activity of caspofungin and micafungin against 1038 yeasts isolates from France by EUCAST reference method E. Dannaoui, O. Lortholary, D. Raoux, D. Hoinard, F. Dromer and the YEASTS Group (Paris, FR) Objectives: To assess the in vitro antifungal susceptibility to caspofungin and micafungin of yeast clinical isolates received at the National Center for Mycoses and Antifungals (CNRMA) from 2003 to 2006. Methods: All isolates were identified according to standard procedures. Susceptibility testing was performed according to the EUCAST broth microdilution reference method. RPMI with 2% glucose was used as test medium with a final inoculum size of 1×105 CFU/mL. Microplates were read spectrophotometrically after 24 h of incubation at 35ºC. MICs were defined as the lowest concentration resulting in 50% or more reduction of growth compared to the drug-free growth control. Quality control strains were included in each set of MIC determination. Results: 1038 yeast isolates belonging to 31 different species were included. Most of the isolates were recovered from blood. Five species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and Cryptococcus neoformans) accounted for 86% of all isolates. Overall, there was a positive correlation between MICs of both drugs. C. albicans, C. glabrata, and C. tropicalis, were the most susceptible species with Gmean MIC of <1 and <0.1 mg/mL for caspofungin and micafungin, respectively. Among other Candida spp. C. parapsilosis, C. guilliermondii, and C. fermentati showed the highest MICs. No activity was found against C. neoformans with Gmean MIC of >9 mg/mL for both echinocandins. Overall, micafungin showed lower MICs than caspofungin.

C. albicans C. glabrata C. parapsilosis C. tropicalis C. guilliermondii C. krusei Cr. neoformans

MIC (mg/mL) Caspofungin Na Range

MIC50

MIC90

GMICb

Micafungin Na Range

MIC50

MIC90

GMICb

404 157 109 62 27 21 158

0.5 0.5 2 0.5 2 1 8

0.5 1 2 1 2 2 16

0.38 0.51 1.57 0.60 1.32 1.10 9.59

161 60 49 23 13 13 64

0.03 0.03 1 0.06 1 0.25 16

0.03 0.03 2 0.06 1 0.25 16

0.027 0.033 1.29 0.052 0.77 0.26 9.62

0.125−4 0.25−8 0.5−4 0.25−2 0.06−2 0.5−8 1−16

0.015−1 0.015−8 0.25−2 0.03–0.125 0.25−1 0.125−4 2−16

Objective: Invasive Fusarium infection is an emerging opportunistic mycosis associated with high mortality. Its diagnosis is difficult due to non-specific signs and symptoms and similarity in tissue morphology with Aspergillus species. The aim of this study was to develop a sensitive and specific diagnostic procedure based on amplification of Fusarium oxysporum DNA by nested (n) PCR and to compare the results with (1-3)-b-D-Glucan detection in serum samples obtained from experimentally infected mice. Methods: Sixty mice immunosuppressed with intraperitoneal injections of cyclophosphamide were infected intravenously with a dose of 1×107 F. oxysporum conidia/mouse. Six mice were sacrificed every day postinfection. Blood specimens were collected by cardiac puncture. One hundred mL of blood was used for culture and the rest was processed for separation of serum. The lung homogenate was cultured and was also used for direct microscopic examination. The genomic DNA from the reference strains of F. oxysporum and 9 other fungi including Fusarium solani, Aspergillus fumigatus, A. flavus and A. terreus was isolated and used as template for nPCR specificity. The species-specific primers were derived from the internally transcribed spacer (ITS)-1 and ITS-2 regions of rDNA and their specificity was confirmed by BLAST searches. The DNA from serum specimens was extracted using standard procedures. The PCR amplicons were detected by agarose gel electrophoresis. The (1-3)-b-D-Glucan was detected by Fungitell (Cape Cod Inc. E. Falmouth, MA, USA). Results: The nPCR was specific for F. oxysporum and detected nearly 440 fg of Fusarium DNA which is roughly equivalent to 10 genome copies. Of the 60 serum samples tested, 55 (92%) were positive for (1-3)-beta-D-Glucan and 47 (78%) were positive for F. oxysporum DNA. The lung homogenates of all the infected animals yielded F. oxysporum in culture. The fungus was also discernible in KOH-calcofluor mount of 40 (67%) of the animals. Conclusions: A sensitive and species-specific nPCR assay has been developed for the detection of F. oxysporum DNA in serum samples of experimentally-infected mice. During a follow-up of 14 days postinfection, 92% and 78% of serum samples were positive for (1-3)-bD-Glucan and F. oxysporum DNA, respectively. The data suggest that detection of F. oxysporum DNA by nPCR combined with (1-3)-b-DGlucan may help in early diagnosis of invasive fusariosis. Supported by KURA Grant MI 04/02. P1976 Comparative evaluation of Aspergillus terreus DNA and galactomannan in serum and bronchoalveolar lavage of intravenously infected mice

Table 1. MIC values for species with at least 10 tested isolates Species

P1975 Detection of Fusarium oxysporum DNA and (1-3)-b-D-glucan in serum specimens of experimentally infected mice

a Number of isolates tested. b Geometric mean MIC.

Conclusions: Caspofungin and micafungin exhibited good in vitro activity by EUCAST method in RPMI medium against most clinical isolates of Candida spp. Although micafungin showed lower MIC values, there was a positive correlation between MICs of both echinocandins.

S. Ahmad, A. Theyyathel, Z. Khan (Kuwait, KW) Objective: Invasive aspergillosis is a growing problem in immunocompromised individuals. Although Aspergillus fumigatus is the principal aetiologic agent of the disease, infections caused by Aspergillus terreus have shown an increased occurrence in recent years. Additionally, A. terreus is more resistant to amphotericin B and is probably also associated with higher mortality in humans. Therefore, an early diagnosis is imperative for reduced mortality and better prognosis. The aim of this study was to develop sensitive and specific diagnosis of invasive aspergillosis by detecting A. terreus-specific DNA by nested PCR (nPCR) in serum and bronchoalveolar lavage (BAL) specimens of intravenously infected mice and to compare the results with galactomannan (GM) levels. Methods: Sixty BALB/c mice, immunosuppressed with four intraperitoneal injections of cyclophosphamide (200 mg/kg) on day −1, 0, +1, +3 were infected intravenously with 1×106 conidia of A. terreus. The mice were sacrificed on day 1, 3, 5, 7 and 9 post-infection in groups of twelve each and their BAL, blood, and lungs were cultured. The A. terreus-specific DNA and GM in serum and BAL were detected by