Antifungal susceptibility testing of Candida spp. by relative growth measurement at single concentrations of antifungal agents.

The relative growth (percentage of growth relative to control growth) of 496 isolates representing six Candida species was assessed as a means of determining in vitro susceptibilities of the isolates in microdilution plate wells containing single concentrations of each of seven antifungal agents. The relative growth data were highly reproducible. With flucytosine and amorolfine they correlated well with MICs, but for an azole antifungal agent, terconazole, they did not correlate with MICs. Distributions of relative growth percentages for different Candida spp. showed significant differences in species susceptibility to individual agents. For example, C. albicans was less susceptible than the other species to amorolfine; C. parapsilosis isolates were particularly susceptible to terbinafine; and C. glabrata, C. guilliermondii, and C. krusei isolates were less susceptible than C. albicans to fluconazole and ketoconazole but equally susceptible as or more susceptible than C. albicans to itraconazole. Differential patterns of susceptibility to individual azole antifungal agents were noted for some individual strains as well as for Candida spp.     

Effects of incubation temperature, inoculum size, and medium on agreement of macro- and microdilution broth susceptibility test results for yeasts.

We examined the effects of temperature and inoculum on the agreement of macro- and microdilution broth MICs of five antifungal agents against six isolates of Candida species or Torulopsis glabrata. Incubation temperature affected results with amphotericin B, flucytosine, fluconazole, and SCH 39304, producing better agreement at 35 degrees C than at 37 degrees C. Agreement between methods was better with an inoculum size of 10(2) than with one of 10(4) yeast cells per ml in testing fluconazole or SCH 39304, and the discrepancies seen with a higher incubation temperature and a larger inoculum appeared to be additive. However, inoculum size did not seem to affect agreement between methods in testing amphotericin B, flucytosine, or ketoconazole. Regardless of test conditions, macrodilution broth MICs of amphotericin B for different isolates were strikingly higher than microdilution test MICs, with mean differences being greater than ninefold under some test conditions. We conclude that for most currently available antifungal agents, an incubation temperature of 35 degrees C and a starting yeast inoculum of less than 10(4) cells per ml improve the agreement between macro- and microdilution broth tests.     

Susceptibility of Candida albicans to flucytosine when tested in different formulations of yeast nitrogen base broth

The influence of various formulations of yeast nitrogen base broth and temperature on the flucytosine sensitivity of Candida albicans was examined. Each of 15 isolates was tested concurrently in yeast nitrogen base from two different suppliers, with or without supplemental dextrose and L-asparagine, and with incubation at either 30 degrees C or 37 degrees C. Observed differences were related to media source, media supplementation, and temperature of incubation. For all three variables, higher endpoint results were associated with greater rates of yeast growth as reflected by turbidity after 24 hr of incubation. Despite these differences, their magnitudes were sufficiently small so that variations in yeast nitrogen base formulation appear not to be an important source of imprecision in susceptibility testing of yeasts.     

Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation.

The technical parameters for antifungal susceptibility testing with Candida species were reexamined to determine the optimal conditions for testing with semiautomated preparations of broth microdilution cultures, automated spectrophotometric readings of the cultures, and dose-response and endpoint determinations by means of a computer spreadsheet. Tests were based on proposed standard method M27P of the National Committee for Clinical Laboratory Standards for antifungal agents. RPMI 1640 broth with extra glucose to a final concentration of 2% gave higher and more reproducible drug-free control readings without affecting susceptibility endpoint readings. An inoculum of 8 x 10(4) yeasts per ml prepared from a carbon-limiting broth culture without further standardization was found to give optimal control readings after 48 h of incubation at 37 degrees C. For flucytosine, fluconazole, itraconazole, and ketoconazole, endpoints based on 50% growth inhibition (50% inhibitory concentration) gave the minimum variation with inoculum size and the fewest endpoint differences with RPMI 1640 medium obtained from two different suppliers. The 50% inhibitory concentration was also the optimal endpoint for fluconazole and ketoconazole susceptibilities in comparison with broth macrodilution MICs determined by the method of the National Committee for Clinical Laboratory Standards. Intralaboratory reproducibility was determined by retrospective analysis of replicate results for isolates retested at random over a 2-year period. This approach showed less favorable reproducibility than has been reported from purpose-designed, prospective antifungal susceptibility studies, but it may better reflect real-life test reproducibility. Susceptibility data for 616 clinical isolates of yeasts, representing 16 Candida and Saccharomyces spp., confirmed the tendency of Candida lusitaniae isolates to show relatively low susceptibilities to amphotericin B, the tendency of Candida krusei isolates to show low flucytosine and fluconazole susceptibilities, and the presence of some isolates in the species Candida albicans, Candida glabrata, and Candida tropicalis with low susceptibilities to azole derivative antifungal agents. The study demonstrates the value of automation and standardization in all stages of yeast susceptibility testing, from plate preparation to data analysis.     

Anticandidal activities of terconazole, a broad-spectrum antimycotic.

Terconazole is a new triazole ketal derivative with broad-spectrum in vitro and in vivo antifungal activities. This study further characterizes the effects of terconazole in vitro on yeast cell growth, viability, and morphology. Terconazole inhibited the growth of Candida albicans ATCC 44859 in a concentration-related manner, but with modest effects noted at levels from 10(-8) to 10(-5) M when the yeast was grown on media favoring the cell form. The inhibitory potency of terconazole on yeast cell viability varied with the strain and species of Candida tested. The susceptibility of C. albicans ATCC 44859 to terconazole was markedly enhanced when the yeast was grown on Eagle minimum essential medium, which favors mycelium formation. The effects of terconazole on the morphology of yeast cells (grown on Eagle minimum essential medium) were shown by phase-contrast and electron microscopy. There is a progression of changes, from loss of mycelia formation at 10(-8) M terconazole through complete necrosis at 10(-4) M.     

Susceptibilities of Candida spp. to antifungal agents visualized by two-dimensional scatterplots of relative growth.

The growth of 811 clinical yeast isolates in the presence of single concentrations of antifungal agents was measured spectrophotometrically and expressed as a percentage of growth in inhibitor-free control cultures. Two-dimensional scatterplots of the relative growth data allowed for the simple visual determination of some susceptibility trends, including correlations in relative growth between different agents and in relative susceptibilities between different yeast species. A positive susceptibility correlation was found for relative growth results with the azole antifungal agents fluconazole, itraconazole, and ketoconazole for 504 Candida albicans isolates. The relative growth scatterplots for fluconazole versus itraconazole showed that 50 (9.9%) of 504 C. albicans isolates were outliers with respect to the 95% confidence limits for a line of correlated relative growth established with an initial test panel of 59 isolates of this species. The outlying isolates were relatively less susceptible to fluconazole than to itraconazole under the conditions of the test. Most of the outliers were received in 1993 and 1994; only 3.9% of the isolates received in 1991 and 1992 and 1.7% of the isolates received before 1991 showed this differential susceptibility. In addition, most of the outliers came from patients with human immunodeficiency virus infections. The relative growth scatterplots confirmed the known high susceptibility of most Candida parapsilosis isolates to both fluconazole and itraconazole and the specifically low susceptibility of Candida krusei isolates to fluconazole. The scatterplots also illustrated a tendency towards lower (and correlative) relative growth among oral isolates obtained from AIDS patients who responded to azole antifungal treatment than among isolates from clinical nonresponders.     

Effect of the growth medium on the in vitro antifungal activity of micafungin (FK-463) against clinical isolates of Candida dubliniensis.

Micafungin (FK-463), a member of the new candin family of antifungal agents, was highly active against clinical isolates of Candida albicans and Candida dubliniensis. The in vitro activity of micafungin suggested that it was more potent than fluconazole, flucytosine, amphotericin B or voriconazole against C. albicans, and comparable or moderately less effective against C. dubliniensis isolates when high-resolution medium (HR) was used. Lower MICs of micafungin were recorded when RPMI 2% or AM3 2% media were used, indicating an influence of the growth medium on the MIC.     

Effect of pH on in vitro susceptibility of Candida glabrata and Candida albicans to 11 antifungal agents and implications for clinical use

The treatment of vulvovaginal candidiasis (VVC) due to Candida glabrata is challenging, with limited therapeutic options. Unexplained disappointing clinical efficacy has been reported with systemic and topical azole antifungal agents in spite of in vitro susceptibility. Given that the vaginal pH of patients with VVC is unchanged at 4 to 4.5, we studied the effect of pH on the in vitro activity of 11 antifungal agents against 40 C. glabrata isolates and compared activity against 15 fluconazole-sensitive and 10 reduced-fluconazole-susceptibility C. albicans strains. In vitro susceptibility to flucytosine, fluconazole, voriconazole, posaconazole, itraconazole, ketoconazole, clotrimazole, miconazole, ciclopirox olamine, amphotericin B, and caspofungin was determined using the CLSI method for yeast susceptibility testing. Test media were buffered to pHs of 7, 6, 5, and 4. Under conditions of reduced pH, C. glabrata isolates remained susceptible to caspofungin and flucytosine; however, there was a dramatic increase in the MIC(90) for amphotericin B and every azole drug tested. Although susceptible to other azole drugs tested at pH 7, C. albicans strains with reduced fluconazole susceptibility also demonstrated reduced susceptibility to amphotericin B and all azoles at pH 4. In contrast, fluconazole-sensitive C. albicans isolates remained susceptible at low pH to azoles, in keeping with clinical observations. In selecting agents for treatment of recurrent C. glabrata vaginitis, clinicians should recognize the limitations of in vitro susceptibility testing utilizing pH 7.0.     

In vitro pharmacodynamic characteristics of flucytosine determined by time-kill methods

Two Candida albicans isolates, three non-albicans Candida isolates (Candida glabrata, Candida krusei, and Candida tropicalis), and one Cryptococcus neoformans isolate were evaluated by time-kill methods to characterize the relationship of flucytosine concentrations to antifungal activity and the duration of the post-antifungal effect (PAE). Against Candida and Cryptococcusisolates, flucytosine at concentrations > 1 x MIC exhibited fungistatic ( 5 h), suggests lower daily dosing may possible without loss of antifungal efficacy.     

Effect of antifungal agents on the binding of Candida albicans to immobilized amino acids and bovine serum albumin.

In this study the effects of different antifungal agents on the binding of Candida albicans yeast cells to different supports were examined. Pre-treatment with amphotericin B or dithiothreitol (DTT) severely reduced the ability of C. albicans yeasts to bind to plastic, while the effects of pre-treatment with fluconazole, ketoconazole or flucytosine were less marked. Both DTT and amphotericin B reduced the binding of yeasts to bovine serum albumin (BSA) and amino acids at low concentrations, while the other antifungal agents were effective at concentrations several-fold higher than their MICs. These data suggest that DTT and amphotericin B affect the yeast cell wall components, and alter both hydrophobic interactions with plastic, and the more specific interactions with BSA and amino acids. By contrast, the effect of the azoles and flucytosine appears to be largely restricted to hydrophobic interactions.     

Effect of fluconazole on viability of Candida albicans over extended periods of time.

The treatment of chronic mycoses may expose the infecting organisms to antimicrobial agents for extended periods of time. It is possible that an azole antifungal drug such as fluconazole, with primarily fungistatic activity in standard in vitro susceptibility tests, might be able to damage the fungal cells and reduce their viability over prolonged incubations under nonproliferating conditions. To test this possibility, Candida albicans yeast cells were exposed to various concentrations of fluconazole in RPMI 1640 tissue culture medium for 4 h at 37 degrees C, washed free of the drug, and then incubated at 37 degrees C for a 28-day period; enumeration of the remaining CFU at various times during this period revealed no increased loss of viability for the fluconazole-exposed organisms. However, when fluconazole was added to the organisms maintained in distilled water (with or without pretreatment with the drug), a marked reduction of viability was found. At 14 days of incubation with two strains of C. albicans, negative cultures were found for 7 of 10 and 10 of 11 samples, respectively, containing 1.0 microgram of fluconazole per ml versus 0 of 10 and 1 of 11 control samples (P of < 0.01 and 0.001, respectively). The effect of fluconazole on fungal viability under these conditions became noticeable at approximately 7 days and was greater when the samples were incubated at 37 degrees C rather than 25 degrees C. These findings suggest that fluconazole may have fungicidal effects on fungal cells during prolonged exposures under conditions in which the organisms are prevented from proliferating by lack of nutrients.     

In vitro activities of isavuconazole and other antifungal agents against Candida bloodstream isolates

Isavuconazole is the active component of the new azole antifungal agent BAL8557, which is entering phase III clinical development. This study was conducted to compare the in vitro activities of isavuconazole and five other antifungal agents against 296 Candida isolates that were recovered consecutively from blood cultures between 1995 and 2004 at a tertiary care university hospital. Microdilution testing was done in accordance with CLSI (formerly NCCLS) guideline M27-A2 in RPMI-1640 MOPS (morpholinepropanesulfonic acid) broth. The antifungal agents tested were amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and isavuconazole. C. albicans was the most common species, representing 57.1% of all isolates. There was no trend found in favor of non-Candida albicans species over time. In terms of MIC(50)s, isavuconazole was more active (0.004 mg/liter) than amphotericin B (0.5 mg/liter), itraconazole (0.008 mg/liter), voriconazole (0.03 mg/liter), flucytosine (0.125 mg/liter), and fluconazole (8 mg/liter). For isavuconazole, MIC(50)s/MIC(90)s ranged from 000.2/0.004 mg/liter for C. albicans to 0.25/0.5 mg/liter for C. glabrata. Two percent of isolates (C. glabrata and C. krusei) were resistant to fluconazole; C. albicans strains resistant to fluconazole were not detected. There were only two isolates with MICs for isavuconazole that were >0.5 mg/liter: both were C. glabrata isolates, and the MICs were 2 and 4 mg/liter, respectively. In conclusion, isavuconazole is highly active against Candida bloodstream isolates, including fluconazole-resistant strains. It was more active than itraconazole and voriconazole against C. albicans and C. glabrata and appears to be a promising agent against systemic Candida infections.     

Histone deacetylase inhibitors enhance Candida albicans sensitivity to azoles and related antifungals: correlation with reduction in CDR and ERG upregulation

Histone acetylation and deacetylation play important roles in eukaryotic gene regulation. Several histone deacetylase (HDA) inhibitors have been characterized, including trichostatin A (TSA), apicidin, and sodium butyrate. We tested their effects on Candida albicans in vitro growth, heat sensitivity, and germ tube formation; minimal effects were observed. However, there was a dramatic effect of TSA on C. albicans sensitivity to the azoles fluconazole, itraconazole, and miconazole. Similar effects were observed with other HDA inhibitors and with the antifungals terbinafine and fenpropimorph, which target, as do azoles, enzymes in the ergosterol biosynthetic pathway. In contrast, HDA inhibitors had minimal effect on the activities of amphotericin B, flucytosine, and echinocandin, which have unrelated targets. Specifically, addition of 3 micro g of TSA/ml lowered the itraconazole MIC for five susceptible C. albicans isolates an average of 2.7-fold at 24 h, but this increased to >200-fold at 48 h. Thus, the primary effect of TSA was a reduction in azole trailing. TSA also enhanced itraconazole activity against Candida parapsilosis and Candida tropicalis but had no effect with four less related yeast species. To examine the molecular basis for these effects, we studied expression of ERG genes (encoding azole and terbinafine targets) and CDR/MDR1 genes (encoding multidrug transporters) in C. albicans cells treated with fluconazole or terbinafine with or without TSA. Both antifungals induced to various levels the expression of ERG1, ERG11, CDR1, and CDR2; addition of TSA reduced this upregulation 50 to 100%. This most likely explains the inhibition of azole and terbinafine trailing by TSA and, more generally, provides evidence that trailing is mediated by upregulation of target enzymes and multidrug transporters.     

Evaluation of amphotericin B and flucytosine in combination against Candida albicans and Cryptococcus neoformans using time-kill methodology.

In this study, time-kill methods were used to evaluate the antifungal activity of amphotericin B and flucytosine, alone and in combination, against six isolates of Candida albicans and Cryptococcus neoformans. Five regimens were tested against each isolate: (1) flucytosine, (2) low-dose amphotericin B, (3) high-dose amphotericin B, (4) low-dose amphotericin B plus flucytosine, and (5) high-dose amphotericin B plus flucytosine. Low-dose amphotericin B and flucytosine, administered alone and simultaneously, demonstrated fungistatic activity against all sample isolates except C. albicans 90028, in which fungicidal activity was detected with the combination. High-dose amphotericin B, alone and in combination, resulted in a rapid fungicidal effect in all isolates. In both the low and high-dose combinations, indifferent activity was demonstrated against all tested isolates. By virtue of the absence of an antagonistic interaction between these two agents, complementary pharmacokinetic profiles, and non-overlapping toxicities, continued clinical use of these agents in combination may be considered.     

Rapid identification of the Candida species from direct blood cultures by CHROMagar Candida

We evaluated the ability of CHROMagar Candida to identify Candida species isolated directly from blood cultures. A total of 50 clinical isolates of Candida were incubated at 35 degrees C, and once growth was established, an aliquot of each was plated onto CHROMagar Candida medium. A control specimen was plated directly from Sabouraud's dextrose agar. Following incubation at 30 degrees C, all yeast isolates were identified by colony morphology and colour. We were able to identify all isolates of C. albicans (n = 20), C. tropicalis (n = 14), C. glabrata (n = 6), and C. krusei (n = 5), which were isolated from blood or from control cultures. This study demonstrated that CHROMagar Candida reliably isolated and identified yeast taken directly from blood cultures. We conclude that this rapid and easy method of identifying Candida species will enable clinicians to quickly choose the appropriate antifungal agent. This should decrease patient morbidity and mortality.     

Susceptibility of clinical isolates of Candida spp. to terconazole and other azole antifungal agents

Terconazole is a triazole ketal derivative with potent, broad-spectrum antifungal activity. We investigated the in vitro activity of terconazole, miconazole, and clotrimazole, against 94 clinical isolates of Candida spp.: C. albicans (n = 68), C. tropicalis (n = 18), and C. parapsilosis (n = 8). In vitro susceptibility testing was performed using a broth microdilution method. The minimal inhibitory concentrations of terconazole were less than those of miconazole against C. albicans and C. parapsilosis but higher against C. tropicalis. Terconazole was more active than clotrimazole against C. parapsilosis and less active against C. albicans and C. tropicalis. Terconazole inhibited the uptake of 14C-labeled glucose, leucine, and hypoxanthine into C. albicans and caused the rapid release of intracellular K+. Based on these studies, terconazole has promising anticandidal activity and warrants further in vitro and in vivo investigation.     

Selective inhibition of 14 alpha-desmethyl sterol synthesis in Candida albicans by terconazole, a new triazole antimycotic

Terconazole, a new broad spectrum antimycotic triazole derivative, has been shown to have potent activity against Candida albicans in vitro and to be effective in animal models of yeast infections. The present study explored a possible mechanism of anticandidal activity of terconazole. The compound inhibited production of 14 alpha-desmethyl sterols (e.g. ergosterol) in C. albicans at concentrations (IC50 = 3-6 x 10(-9) M) lower than those inhibiting the in-vitro growth of the yeast. There was concomitant accumulation of methylated sterols, (e.g. lanosterol), which are considered detrimental to normal yeast cell membrane function. Terconazole stimulated incorporation of 14C-acetate into triglycerides, but had no other effect on C. albicans lipid metabolism. At concentrations greater than or equal to 10(-6)M terconazole inhibited the oxidation of 14C-acetate into 14CO2 in C. albicans although the mechanism for this effect remains unclear. These data indicate that terconazole is a specific inhibitor of yeast C-14 desmethyl sterol production in C. albicans. Furthermore, terconazole reduced cytochrome P-450 levels in yeast microsomes at concentrations 10,000-fold below those at which it showed effects on rabbit liver microsomes. These data indicate a species specificity for the biochemical actions of terconazole. The C-14 alpha-desmethylase system in yeast cell membranes is cytochrome P-450 associated. Thus, terconazole, was a potent inhibitor of C-14 desmethyl sterol synthesis. This effect could contribute to the anticandidal activity of the drug.     

Activities and ultrastructural effects of antifungal combinations against simulated Candida endocardial vegetations

In vitro pharmacodynamic model (PDM) simulation of serum antifungal concentrations may predict the value of combination antifungal regimens against Candida sp. endocarditis. We investigated the effects of combinations of flucytosine (5FC), micafungin (Mica), and voriconazole (Vor) against Candida-infected human platelet-fibrin clots, used as simulated endocardial vegetations (SEVs). Single clinical bloodstream isolates of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis were used. All four isolates were susceptible to 5FC, while C. glabrata was resistant to Vor and C. tropicalis had a paradoxical resistance phenotype to Mica. The SEVs were prepared with an initial inoculum of 1 x 10(6) CFU/g of SEV and added to a PDM, which utilized yeast nitrogen broth-2% glucose and incubation at 35 degrees C and simulated antifungal pharmacokinetic profiles. Fungal densities in the SEVs were determined in quadruplicate over 72 h. Scanning electron microscopy (SEM) was used to evaluate treatment and control SEVs. Vor was the least active single agent against all Candida spp. except for C. parapsilosis, where it was comparable to Mica. In contrast, 5FC was the most active against all Candida spp. except for C. tropicalis, where it was comparable to Mica. The combination of 5FC plus Vor was superior to either agent alone against C. parapsilosis. The combination of Vor plus Mica was inferior to the use of Mica alone against C. tropicalis. The triple combination of 5FC plus Vor plus Mica was no better than single or dual agents against any of the Candida spp. The ultrastructural features of infected SEVs were unique for each Candida sp., with C. parapsilosis in particular manifesting friable biofilm clusters. In general, 5FC and Mica were superior in their rates and extents of fungal burden reduction compared to Vor against Candida-infected SEVs. Evaluation of 5FC and Mica in animal models of Candida endocarditis is warranted.     

The European Confederation of Medical Mycology (ECMM) survey of candidaemia in Italy: in vitro susceptibility of 375 Candida albicans isolates and biofilm production

OBJECTIVES: To investigate the in vitro antifungal susceptibility pattern of 375 Candida albicans bloodstream isolates recovered during the European Confederation of Medical Mycology survey of candidaemia performed in Lombardia, Italy and to test the ability to form biofilm. METHODS: In vitro susceptibility to flucytosine, fluconazole, itraconazole, posaconazole, voriconazole and caspofungin was performed by broth microdilution following the NCCLS guidelines. Biofilm production was measured using the XTT reduction assay in 59 isolates selected as representative of different patterns of susceptibility to flucytosine and azoles. RESULTS: MICs (mg/L) at which 90% of the strains were inhibited were < or =0.25 for flucytosine, 0.25 for caspofungin, 4 for fluconazole and 0.06 for itraconazole, voriconazole and posaconazole. Flucytosine resistance was detected in five isolates and was associated with serotype B in 2/29 and serotype A in 3/346. Resistance to fluconazole was detected in 10 isolates; nine of these exhibited reduced susceptibility to the other azoles. Among the 10 patients with fluconazole-resistant C. albicans bloodstream infection, only one, an AIDS patient, had been previously treated with fluconazole. Biofilm production was observed in 23 isolates (39%) and was significantly associated with serotype B. No relationship was detected with the pattern of antifungal susceptibility. CONCLUSIONS: Resistance is uncommon in C. albicans isolates recovered from blood cultures, while biofilm production is a relatively frequent event. Periodic surveillance is warranted to monitor the incidence of in vitro antifungal resistance as well as of biofilm production.