Francesco Barchiesi

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)∗

Antifungal susceptibility tests are performed on fungi that cause disease, especially if they belong to a species exhibiting resistance to commonly used antifungal agents. Antifungal susceptibility testing is also important for resistance surveillance, for epidemiological studies and for comparing the in-vitro activity of new and existing agents. Dilution methods are used to establish the MICs of antimicrobial agents. These are the reference methods for antimicrobial susceptibility testing, and are used mainly to establish the activity of a new antifungal agent, to confirm the susceptibility of organisms that give equivocal results in routine tests, and to determine the susceptibility of fungi where routine dilution tests may be unreliable. Fungi are tested for their ability to produce visible growth in microdilution plate wells containing broth culture media and serial dilutions of the antifungal agents (broth microdilution). The MIC is defined as the lowest concentration (in mg ⁄ L) of an antifungal agent that inhibits the growth of a fungus. The MIC provides information concerning the susceptibility or resistance of an organism to the antifungal agent and can help in making correct treatment decisions. The method described in this document is intended for testing the susceptibility of yeasts that cause clinically significant infections (primarily Candida spp.). The method encompasses only those yeasts that are able to ferment glucose. Thus, the susceptibility of non-fermentative yeasts, e.g., Cryptococcus neoformans, cannot be determined by the current procedure, and the method is not suitable for testing the yeast forms of dimorphic fungi.

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts

Antifungal susceptibility tests are performed on fungi that cause disease, especially if they belong to a species exhibiting resistance to commonly used antifungal agents. Antifungal susceptibility testing is also important for resistance surveillance, for epidemiological studies and for comparing the in-vitro activity of new and existing agents. Dilution methods are used to establish the MICs of antimicrobial agents. These are the reference methods for antimicrobial susceptibility testing, and are used mainly to establish the activity of a new antifungal agent, to confirm the susceptibility of organisms that give equivocal results in routine tests, and to determine the susceptibility of fungi where routine dilution tests may be unreliable. Fungi are tested for their ability to produce visible growth in microdilution plate wells containing broth culture media and serial dilutions of the antifungal agents (broth microdilution). The MIC is defined as the lowest concentration (in mg ⁄ L) of an antifungal agent that inhibits the growth of a fungus. The MIC provides information concerning the susceptibility or resistance of an organism to the antifungal agent and can help in making correct treatment decisions. The method described in this document is intended for testing the susceptibility of yeasts that cause clinically significant infections (primarily Candida spp.). The method encompasses only those yeasts that are able to ferment glucose. Thus, the susceptibility of non-fermentative yeasts, e.g., Cryptococcus neoformans, cannot be determined by the current procedure, and the method is not suitable for testing the yeast forms of dimorphic fungi.

International and Multicenter Comparison of EUCAST and CLSI M27-A2 Broth Microdilution Methods for Testing Susceptibilities of Candida spp. to Fluconazole, Itraconazole, Posaconazole, and Voriconazole

ABSTRACT The aim of this study was to compare MICs of fluconazole, itraconazole, posaconazole, and voriconazole obtained by the European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI (formerly NCCLS) methods in each of six centers for 15 Candida albicans (5 fluconazole-resistant and 4 susceptible-dose-dependent [S-DD] isolates), 10 C. dubliniensis, 7 C. glabrata (2 fluconazole-resistant isolates), 5 C. guilliermondii (2 fluconazole-resistant isolates), 10 C. krusei, 9 C. lusitaniae, 10 C. parapsilosis, and 5 C. tropicalis (1 fluconazole-resistant isolate) isolates. CLSI MICs were obtained visually at 24 and 48 h and spectrophotometric EUCAST MICs at 24 h. The agreement (within a 3-dilution range) between the methods was species, drug, and incubation time dependent and due to lower EUCAST than CLSI MICs: overall, 94 to 95% with fluconazole and voriconazole and 90 to 91% with posaconazole and itraconazole when EUCAST MICs were compared against 24-h CLSI results. The agreement was lower (85 to 94%) against 48-h CLSI endpoints. The overall interlaboratory reproducibility by each method was ≥92%. When the comparison was based on CLSI breakpoint categorization, the agreement was 68 to 76% for three of the four species that included fluconazole-resistant and S-DD isolates; 9% very major discrepancies (≤8 μg/ml versus ≥64 μg/ml) were observed among fluconazole-resistant isolates and 50% with voriconazole (≤1 μg/ml versus ≥4 μg/ml). Similar results were observed with itraconazole for seven of the eight species evaluated (28 to 77% categorical agreement). Posaconazole EUCAST MICs were also substantially lower than CLSI MIC modes (0.008 to 1 μg/ml versus 1 to ≥8 μg/ml) for some of these isolates. Therefore, the CLSI breakpoints should not be used to interpret EUCAST MIC data.

Experimental Induction of Fluconazole Resistance in Candida tropicalis ATCC 750

ABSTRACT Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans. UnlikeC. albicans, which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis, an FLC-susceptible strain (ATCC 750) (MIC = 1.0 μg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 μg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 μg/ml for strains grown in medium with 8.0, 32, and 128 μg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 μg/ml) and terbinafine (MIC > 512 μg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 μg/ml reverted to low MICs (1.0 μg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 μg/ml) (128 R) reverted to 16 μg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis.

Effects of Caspofungin against Candida guilliermondii and Candida parapsilosis

ABSTRACT The in vitro activity of caspofungin (CAS) was investigated against 28 yeast isolates belonging to Candida albicans (n = 5), Candida guilliermondii (n = 10), and Candida parapsilosis (n = 13). CAS MICs obtained by broth dilution and Etest methods clearly showed a rank order of susceptibility to the echinocandin compound with C. albicans > C. parapsilosis > C. guilliermondii. Similarly, time-kill assays performed on selected isolates showed that CAS was fungistatic against C. albicans and C. parapsilosis, while it did not exert any activity against C. guilliermondii. In a murine model of systemic candidiasis, CAS given at doses as low as 1 mg/kg of body weight/day was effective at reducing the kidney burden of mice infected with either C. albicans or C. guilliermondii isolates. Depending on the isolate tested, mice infected with C. parapsilosis responded to CAS given at 1 and/or 5 mg/kg/day. However, the overall CFU reduction for C. guilliermondii and C. parapsilosis was approximately 100-fold less than that for C. albicans. Our study shows that CAS was active in experimental systemic candidiasis due to C. guilliermondii and C. parapsilosis, but this activity required relatively high drug dosages.

Interactions between Triazoles and Amphotericin B against Cryptococcus neoformans

ABSTRACT The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates ofCryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to ≤2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of C. neoformans 2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of C. neoformans 2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted with nonreplicating cells showed that preexposure to FLC abolished the fungicidal activity of the polyene. However, this apparent antagonism was not observed in vivo. Rather, when the two drugs were used sequentially for the treatment of systemic murine cryptococcosis, a reciprocal potentiation was often observed. Our study shows that (i) the combination of triazoles and AmB is significantly more active than either drug alone against C. neoformans in vitro and (ii) the concomitant or sequential use of FLC and AmB for the treatment of systemic murine cryptococcosis results in a positive interaction.

In Vitro Activities of the New Antifungal Triazole SCH 56592 against Common and Emerging Yeast Pathogens

ABSTRACT A broth microdilution method performed in accordance with the National Committee for Clinical Laboratory Standards guidelines was used to compare the in vitro activity of the new antifungal triazole SCH 56592 (SCH) to that of fluconazole (FLC), itraconazole (ITC), and ketoconazole (KETO) against 257 clinical yeast isolates. They included 220 isolates belonging to 12 different species of Candida, 15 isolates each of Cryptococcus neoformans andSaccharomyces cerevisiae, and seven isolates ofRhodotorula rubra. The MICs of SCH at which 50% (MIC50) and 90% (MIC90) of the isolates were inhibited were 0.06 and 2.0 μg/ml, respectively. In general, SCH was considerably more active than FLC (MIC50 and MIC90 of 1.0 and 64 μg/ml, respectively) and slightly more active than either ITC (MIC50 and MIC90 of 0.25 and 2.0 μg/ml, respectively) and KETO (MIC50 and MIC90 of 0.125 and 4.0 μg/ml, respectively). Our in vitro data suggest that SCH has significant potential for clinical development.

In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics

The in-vitro activity of nisin, a 34-residue peptide produced by several Lactococcus lactis strains, and ranalexin, a 20-residue peptide isolated from the skin of the bullfrog Rana catesbeiana, alone and in combination with amoxycillin, amoxycillin-clavulanate, imipenem, clarithromycin, ciprofloxacin, rifampin and vancomycin was investigated against 40 nosocomial isolates of methicillin-resistant Staphylococcus aureus (MRSA). All isolates were inhibited at concentrations of 1 to 32 microg/ml. Synergy was observed when the peptides were combined with other agents, with the exception of the beta-lactams. Finally, the consecutive exposures to each peptide did not result in selection of stable mutants with decreased susceptibility. Our finding show that nisin and ranalexin are active against MRSA, and that their activity is enhanced when they are combined with several antimicrobial agents.

Candida guilliermondii Fungemia in Patients with Hematologic Malignancies

ABSTRACT The microbiological, clinical, and epidemiological features of most non-Candida albicans Candida species are well known, but much less is known about species such as Candida guilliermondii, an uncommon pathogen causing a variety of deep-seated infections in immunocompromised hosts. To characterize C. guilliermondii fungemia in patients with hematological malignancies and its susceptibility to antifungal drugs, all cases of C. guilliermondii fungemia diagnosed in our department between 1983 and 2005 were retrospectively analyzed and the literature was reviewed. C. guilliermondii caused 29/243 (11.7%) candidemia episodes diagnosed during the study period. Central venous catheters were the documented sources of candidemia in 19/29 episodes (65.5%), and invasive tissue infections were documented in 2 (6.9%). In the remaining eight, the catheter was not removed and the source of the fungemia remained obscure. Seven episodes ended in death, but only one could be attributed to invasive C. guilliermondii infection. Molecular typing data reveal no evidence of common infection sources. Isolates displayed high rates of in vitro susceptibility to amphotericin B (100%), voriconazole (95%), and fluconazole (90%) and lower rates of in vitro susceptibility to flucytosine (86%), itraconazole (76%), and caspofungin (33%). Our literature review confirms that C. guilliermondii is a significantly more frequent cause of candidemia among cancer patients compared with the general hospital population. It accounted for <1% of the total number of Candida bloodstream isolates reported in the articles we reviewed, with higher rates in Europe (1.4%) and Asia (1.8%) compared with North America (0.3%).

Antimicrobial activity of polycationic peptides.

The in vitro activity of six polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated against several clinical isolates of gram-positive and gram-negative aerobic bacteria, yeasts, Pneumocystis carinii and Cryptosporidium parvum, by using microbroth dilution methods. The peptides exhibited different antibacterial activities and rapid time-dependent killing. The gram-negative organisms were more susceptible to buforin II and cecropin P1, whereas buforin II and ranalexin were the most active compounds against the gram-positive strains. Similarly, ranalexin showed the highest activity against Candida spp., whereas magainin II exerted the highest anticryptococcal activity. Finally, the peptides showed high anti-Pneumocystis activity, whereas no compound had strong inhibitory effect on C. parvum.