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ABSTRACTThe CLSI epidemiological cutoff values (ECVs) of antifungal agents are available for various Candidaspp., Aspergillusspp., and the Mucorales. However, those categorical endpoints have not been established for Fusariumspp., mostly due to the difficulties associated with collecting sufficient CLSI MICs for clinical isolates identified according to the currently recommended molecular DNA-PCR-based identification methodologies. CLSI MIC distributions were established for 53 Fusarium dimerumspecies complex (SC), 10 F. fujikuroi, 82 F. proliferatum, 20 F. incarnatum-F. equisetiSC, 226 F. oxysporumSC, 608 F. solaniSC, and 151 F. verticillioidesisolates originating in 17 laboratories (in Argentina, Australia, Brazil, Canada, Europe, Mexico, and the United States). According to the CLSI guidelines for ECV setting, ECVs encompassing ≥97.5% of pooled statistically modeled MIC distributions were as follows: for amphotericin B, 4 μg/ml (F. verticillioides) and 8 μg/ml (F. oxysporumSC and F. solaniSC); for posaconazole, 2 μg/ml (F. verticillioides), 8 μg/ml (F. oxysporumSC), and 32 μg/ml (F. solaniSC); for voriconazole, 4 μg/ml (F. verticillioides), 16 μg/ml (F. oxysporumSC), and 32 μg/ml (F. solaniSC); and for itraconazole, 32 μg/ml (F. oxysporumSC and F. solaniSC). Insufficient data precluded ECV definition for the other species. Although these ECVs could aid in detecting non-wild-type isolates with reduced susceptibility to the agents evaluated, the relationship between molecular mechanisms of resistance (gene mutations) and MICs still needs to be investigated for Fusariumspp.

ABSTRACTThe treatment of onychomycosis has improved considerably over the past several decades following the introduction of the oral antifungals terbinafine and itraconazole. However, these oral agents suffer from certain disadvantages, including drug interactions and potential liver toxicity. Thus, there is a need for new topical agents that are effective against onychomycosis. ME1111 is a novel selective inhibitor of succinate dehydrogenase (complex II) of dermatophyte species, whose small molecular weight enhances its ability to penetrate the nail plate. In this study, we determined the antifungal activity of ME1111 against dermatophyte strains, most of which are known to cause nail infections, as measured by the MIC (n= 400) and the minimum fungicidal concentration (MFC) (n= 300). Additionally, we examined the potential for resistance development in dermatophytes (n= 4) following repeated exposure to ME1111. Our data show that the MIC90of ME1111 against dermatophyte strains was 0.25 μg/ml, which was equivalent to that of the comparators amorolfine and ciclopirox (0.25 and 0.5 μg/ml, respectively). ME1111 was fungicidal at clinically achievable concentrations against dermatophytes, and its MFC90s against Trichophyton rubrumand Trichophyton mentagrophyteswere 8 μg/ml, comparable to those of ciclopirox. Furthermore, ME1111, as well as ciclopirox, did not induce resistance in 4 dermatophytes tested. Our studies show that ME1111 possesses potent antifungal activity and suggest that it has low potential for the development of resistance in dermatophytes.

ABSTRACTClinical breakpoints (CBPs) have not been established for the Mucoralesand any antifungal agent. In lieu of CBPs, epidemiologic cutoff values (ECVs) are proposed for amphotericin B, posaconazole, and itraconazole and four Mucoralesspecies. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) were defined with available pooled CLSI MICs from 14 laboratories (Argentina, Australia, Canada, Europe, India, Mexico, and the United States) as follows: 10 Apophysomyces variabilis, 32 Cunninghamella bertholletiae, 136 Lichtheimia corymbifera, 10 Mucor indicus, 123 M. circinelloides, 19 M. ramosissimus, 349 Rhizopus arrhizus, 146 R. microsporus, 33 Rhizomucor pusillus, and 36 Syncephalastrum racemosumisolates. CLSI broth microdilution MICs were aggregated for the analyses. ECVs comprising ≥95% and ≥97.5% of the modeled populations were as follows: amphotericin B ECVs for L. corymbiferawere 1 and 2 μg/ml, those for M. circinelloideswere 1 and 2 μg/ml, those for R. arrhizuswere 2 and 4 μg/ml, and those for R. microsporuswere 2 and 2 μg/ml, respectively; posaconazole ECVs for L. corymbiferawere 1 and 2, those for M. circinelloideswere 4 and 4, those for R. arrhizuswere 1 and 2, and those for R. microsporuswere 1 and 2, respectively; both itraconazole ECVs for R. arrhizuswere 2 μg/ml. ECVs may aid in detecting emerging resistance or isolates with reduced susceptibility (non-WT MICs) to the agents evaluated.

ABSTRACTEsophageal candidiasis is a frequent cause of morbidity in immunocompromised patients. Isavuconazole is a novel, broad-spectrum antifungal developed for the treatment of opportunistic fungal infections. This phase 2 trial compared the efficacy and safety of three oral dosing regimens of isavuconazole with an oral fluconazole regimen in the primary treatment of uncomplicated esophageal candidiasis. The isavuconazole regimens were as follows: 200 mg on day 1 and then 50 mg once daily (arm A), 400 mg on day 1 and then 400 mg once-weekly (arm B), and 400 mg on day 1 and then 100 mg once daily (arm C). Patients in arm D received fluconazole at 200 mg on day 1 and then 100 mg once daily. The minimum treatment duration was 14 days. The primary endpoint was the rate of endoscopically confirmed clinical response at end of therapy. Safety and tolerability were also assessed. Efficacy was evaluated in 153 of 160 enrolled patients. Overall, 146 (95.4%) achieved endoscopically confirmed clinical success. Each of the isavuconazole regimens was shown to be not inferior to fluconazole, i.e., arm A versus D, −0.5% (95% confidence interval [CI] −10.0 to 9.4), arm B versus D, 3.5% (95% CI, −5.6 to 12.7), and arm C versus D, −0.2% (95% CI, −9.8 to 9.4). The frequency of adverse events was similar in arm A (n= 22; 55%), arm B (n= 18; 45%), and arm D (n= 22; 58%), but higher in arm C (n= 29; 71%). In summary, efficacy and safety of once-daily and once-weekly isavuconazole were comparable with once-daily fluconazole in the primary treatment of uncomplicated esophageal candidiasis.

ABSTRACTThe incidence of superficial dermatophytoses is high in developed countries, and there remains a need for effective topical antifungals. In this study, we evaluated the in vitroantifungal activity of naftifine hydrochloride, the active ingredient in naftifine hydrochloride cream and gel 1% and 2%, against dermatophytes. The MICs and minimum fungicidal concentrations (MFCs) of naftifine hydrochloride against 350 clinical strains, including Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Epidermophyton floccosum, and Microsporum canis, were determined using the CLSI methodology. Subsets from this test panel were subsequently tested in a time-kill assay at 0.125×, 0.25×, 0.5×, and 1× the MFC for each isolate. CFU counts were performed over a period of 48 h of incubation. Additionally, in order to determine the potential for resistance development, six strains were subjected to 15 serial passages in concentrations higher than the MIC for each strain. MICs were determined following each passage. The MIC range against the dermatophyte isolates tested was 0.015 to 1.0 μg/ml, with naftifine hydrochloride being fungicidal against 85% of the Trichophytonspecies. The time-kill assay showed dose-dependent activity, with the greatest reduction in the numbers of CFU corresponding to the highest drug concentration. There was no increase in MIC for any strains following repeated exposure to naftifine hydrochloride. Naftifine hydrochloride demonstrated potent activity against all dermatophytes tested, and none of the isolates within this test panel demonstrated the potential for the development of resistance. Thus, future clinical studies of naftifine hydrochloride against dermatophytes may be warranted for the treatment of superficial dermatophytoses.

ABSTRACTTDT 067 is a novel, carrier-based dosage form of terbinafine in Transfersome (1.5%) formulated for topical delivery of terbinafine to the nail, nail bed, and surrounding tissue. We examined the effects of TDT 067 and conventional terbinafine on the morphology of dermatophytes. Trichophyton rubrumhyphae were exposed to TDT 067 or terbinafine (15 mg/ml) and examined under white light, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Subungual debris from patients treated with TDT 067 in a clinical trial was also examined. Exposure of T. rubrumhyphae to TDT 067 led to rapid and extensive ultrastructural changes. Hyphal distortion was evident as early as 4 h after exposure to TDT 067. After 24 h, there was complete disruption of hyphal structure with few intact hyphae remaining. Exposure to terbinafine resulted in morphological alterations similar to those seen with TDT 067; however, the effects of TDT 067 were more extensive, whereas a portion of hyphae remained intact after 24 h of exposure to terbinafine. Lipid droplets were observed under TEM following 30 min of exposure to TDT 067, which after 24 h had filled the intracellular space. These effects were confirmed in vivoin subungual debris from patients with onychomycosis who received topical treatment with TDT 067. The Transfersome in TDT 067 may potentiate the action of terbinafine by delivering terbinafine more effectively to its site of action inside the fungus. Our in vivodata confirm that TDT 067 can enter fungus in the nail bed of patients with onychomycosis and exert its antifungal effects.

ABSTRACTAlthough clinical breakpoints have not been established for mold testing, epidemiological cutoff values (ECVs) are available for Aspergillusspp. versus the triazoles and caspofungin. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six Aspergillusspp. and amphotericin B. Two sets (CLSI/EUCAST broth microdilution) of available MICs were evaluated: those for A. fumigatus(3,988/833), A. flavus(793/194), A. nidulans(184/69), A. niger(673/140), A. terreus(545/266), and A. versicolor(135/22). Three sets of data were analyzed: (i) CLSI data gathered in eight independent laboratories in Canada, Europe, and the United States; (ii) EUCAST data from a single laboratory; and (iii) the combined CLSI and EUCAST data. ECVs, expressed in μg/ml, that captured 95%, 97.5%, and 99% of the modeled wild-type population (CLSI and combined data) were as follows: for A. fumigatus, 2, 2, and 4; for A. flavus, 2, 4, and 4; for A. nidulans, 4, 4, and 4; for A. niger, 2, 2, and 2; for A. terreus, 4, 4, and 8; and for A. versicolor, 2, 2, and 2. Similar to the case for the triazoles and caspofungin, amphotericin B ECVs may aid in the detection of strains with acquired mechanisms of resistance to this agent.

ABSTRACTBiofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candidabiofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicansand Candida parapsilosisbiofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candidabiofilms. We also explored effects of preincubation of C. albicanscells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candidabiofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candidaisolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candidabiofilms. Preincubation of C. albicanscells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P< 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candidabiofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

ABSTRACTThe most important role of susceptibility testing is to identify potentially resistant isolates for the agent being evaluated. Standard testing guidelines recently have been proposed for antifungal susceptibility testing of filamentous fungi (molds). This collaborative (eight centers) study evaluated further newly proposed guidelines (NCCLS, proposed standard M38-P, 1998) and other testing conditions for antifungal susceptibility testing of Aspergillusspp. to itraconazole and three new triazoles, posaconazole (SCH56592), ravuconazole (BMS-207147), and voriconazole. MICs of itraconazole, posaconazole, ravuconazole, and voriconazole for 15 selected isolates of three species of Aspergillus(A. fumigatus, A. flavus, and A. terreus) with well documented in vitro, clinical, or animal data were determined in each center by using four medium formulations (standard RPMI-1640 [RPMI], RPMI with 2% dextrose, antibiotic medium 3 [M3], and M3 with 2% dextrose) and two criteria of MIC determination (complete [MIC-0s] and prominent [MIC-2s] growth inhibition) at 24, 48, and 72 h. The highest reproducibility (92 to 99%) was seen with the standard RPMI and M3 media. Moreover, the distinction between itraconazole-resistant (MICs of >8 μg/ml for clinically resistant strains) and -susceptible (MICs of 0.03 to 1 μg/ml) isolates, as well as between a voriconazole-resistant laboratory mutant and other isolates (voriconazole MICs of 2 to >8 versus 0.12 to 2 μg/ml), was more consistently evident with the standard RPMI medium and when MIC-0s were determined at 48 h. These results provide further refinement of the testing guidelines for susceptibility testing ofAspergillusspp. and warrant consideration for inclusion in the future NCCLS document M38-A.

Interactions among antineoplastic and antifungal drugs affecting the inhibition of Candida albicans growth are complex functions of the nature of the drugs used in combination, their absolute concentrations, and also their relative concentrations. Studies of drug interactions involving the use of test drugs in fixed concentration ratios can lead to inaccurate conclusions about synergism or antagonism among the drugs. A multifactorial experimental design procedure in which the concentrations of all drugs in test combinations were simultaneously varied has been used to identify and quantify drug interactions. The methods have been applied to combinations of two, three, and four drugs, including antineoplastic drugs, antifungal drugs, and combinations of antineoplastic and antifungal drugs. Results were obtained which allow predictions of effects of combinations and provide maximum effectiveness in growth inhibition with minimum levels of the test drugs.

The effects of combinations of antifungal and antineoplastic drugs on inhibition of the growth of yeasts which commonly infect cancer patients have been analyzed. It was shown that (i) inhibitory drug combinations could be selected in which all drugs were at levels far below their individual MICs; (ii) interactive effects among antineoplastic and antifungal drugs may be very large; (iii) optimum combinations of drugs for inhibition of yeast growth depended upon both the relative and absolute concentrations of the drugs in the mixture; (iv) drug combinations which were effective at low levels in inhibiting one test yeast were also generally effective against other species, but the levels of susceptibilities and, to a lesser extent, the best ratios of drugs in the test combinations varied with species; and (v) to quantitatively evaluate drug interactions, it is necessary to carefully define and control all experimental conditions, absolute and relative concentrations of drugs used, and the organisms tested.

Candida aurishas been shown to have a high risk of skin colonization in hospitalized patients, possibly contributing to nosocomial spread. In a guinea pig skin model, animals were evaluated for clinical appearance, tissue fungal burden, histology, and pharmacokinetics. Oral dosing with 10 mg/kg ibrexafungerp (IBX) reduced the severity of lesions and significantly reduced the C. aurisfungal burden in infected animals compared with untreated controls.

ABSTRACTWe investigated the in vitro activity of voriconazole compared to those of fluconazole and itraconazole against 566 clinical isolates ofCryptococcus neoformansfrom Africa (164) and the United States (402). Isolates were obtained from cerebrospinal fluid (362), blood (139), and miscellaneous sites (65). Voriconazole (MIC at which 90% of the isolates are inhibited [MIC90], 0.12 to 0.25 μg/ml) was more active than either itraconazole (MIC90, 0.5 μg/ml) or fluconazole (MIC90, 8.0 to 16 μg/ml) against both African and U.S. isolates. Isolates inhibited by ≥16 μg of fluconazole per ml were almost all (99%) inhibited by ≤1 μg of voriconazole per ml. These results suggest that voriconazole may be useful in the treatment of cryptococcosis.

Central-line-associated bloodstream infections are increasingly recognized to be associated with intraluminal microbial biofilms, and effective measures for the prevention and treatment of bloodstream infections remain lacking. This report evaluates a new commercially developed antimicrobial catheter lock solution (ACL), containing trimethoprim (5 mg/ml), ethanol (25%), and calcium EDTA (Ca-EDTA) (3%), for activity against bacterial and fungal biofilms, using in vitroand in vivo(rabbit) catheter biofilm models.

ABSTRACTThe triple combination trimethoprim, EDTA, and ethanol (B-Lock), is an antimicrobial lock solution for use in indwelling intravascular catheters to prevent and treat catheter-associated infections. B-Lock demonstrated MICs of ≤0.05% (percentage of solution) against Candidaspp. (n= 125) and 0.003% to 25% against bacterial strains (n= 175). B-Lock was also fungicidal against the majority of the Candidastrains at 6% to 25%. B-Lock demonstrates potential value for the prevention and treatment of catheter-associated infections.