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.     

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.     

Influence of Human Serum on Antifungal Pharmacodynamics with Candida albicans

Antifungal susceptibilities (NCCLS, approved standard M27-A, 1997) were determined for the reference strain ATCC 90028 and 21 clinical isolates of Candida albicans with varying levels of fluconazole susceptibility using RPMI 1640 (RPMI) and 80% fresh human serum-20% RPMI (serum). Sixty-four percent (14 of 22) of the isolates tested demonstrated significant decreases (> or = 4-fold) in fluconazole MICs in the presence of serum, and the remaining eight isolates exhibited no change. Itraconazole and ketoconazole, two highly protein-bound antifungal agents, had MICs in serum that were increased or unchanged for 46% (10 of 22) and 41% (9 of 22) of the isolates, respectively. All 10 isolates tested against an investigational antifungal agent, LY303366, demonstrated significant increases in the MIC required in serum, while differences in amphotericin B MICs in the two media were not observed. Four of 10 isolates tested demonstrated fourfold higher flucytosine MICs in serum than in RPMI. Postantifungal effects (PAFEs) and 24-h kill curves were determined by standard methods for selected isolates. At the MIC, fluconazole, itraconazole, ketoconazole, flucytosine, and LY303366 kill curves and PAFEs in RPMI were similar to those in serum. Isolates of fluconazole-resistant C. albicans required lower MICs in serum than in RPMI, without relative increases in fungal killing or PAFEs. Isolates tested against amphotericin B demonstrated significantly reduced killing and shorter PAFEs in serum than in RPMI without observable changes in MIC. In conclusion, antifungal pharmacodynamics in RPMI did not consistently predict antifungal activity in serum for azoles and amphotericin B. Generally speaking, antifungal agents with high protein binding exhibited some form of reduced activity (MIC, killing, or PAFE) in the presence of serum compared to those with low protein binding.     

Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans. Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.     

Effects of temperature on anti-Candida activities of antifungal antibiotics.

The relative growth (percentage of growth relative to control growth) of 767 Candida isolates representing five species was measured in microcultures at 25 and 37 degrees C. In the presence of 10(-4) M flucytosine, the distribution of relative yeast growth data indicated that Candida albicans isolates were less susceptible at 25 degrees C than at 37 degrees C, while the opposite was found with 4 x 10(-5) M amorolfine for most of the isolates tested. Repetition of the experiments at four different temperatures with 99 C. albicans isolates and five antifungal agents confirmed a direct relationship between growth inhibition and increasing temperature from 25 to 40 degrees C with amphotericin B, flucytosine, and terconazole; a strong inverse relationship between inhibition and temperature with amorolfine; and a weak inverse relationship with terbinafine. However, these relationships were not always noted with other Candida spp.: in particular, the growth of C. glabrata and C. parapsilosis isolates tended to be greater at 37 degrees C than at 25 degrees C in the presence of the azole-derivative antifungal agents itraconazole and terconazole. These findings stress the species-specific individuality of yeast susceptibility to azole antifungal agents. The results with C. albicans and amorolfine and terbinafine accord with their known in vivo efficacy in mycoses involving low-temperature superficial sites and poor activity against mycoses involving deep body sites. The data also reinforce the need for control of experimental variables such as temperature in the design of standardized yeast susceptibility tests.     

Development of resistance in candida isolates from patients receiving prolonged antifungal therapy.

The impact of prolonged antifungal therapy on the development of resistance was examined in 61 patients with oropharyngeal thrush. Fifty-nine patients had symptomatic human immunodeficiency virus infection, one had lung cancer, and one had metastatic prostate cancer. Cultures of pharyngeal samples from all patients were positive for yeasts and included 57 (93.4%) Candida albicans, 3 (4.9%) Candida glabrata, and 1 (1.6%) Candida krusii. Of 61 patients, 32 (52.5%) were receiving or had recently received antifungal therapy. Clotrimazole was the most commonly prescribed azole, followed by ketoconazole and fluconazole. Two patients had received amphotericin B therapy and one had received flucytosine. The duration of therapy with clotrimazole, ketoconazole, and fluconazole ranged from 3 to 240, 14 to 44, and 7 to 138 days, respectively. There was no overall difference in the susceptibilities of the clinical isolates from treated and untreated patients to amphotericin B, nystatin, flucytosine, clotrimazole, ketoconazole, and fluconazole. A.C. albicans isolate from one patient who had clinically failed on ketoconazole, fluconazole, and amphotericin B was resistant to these drugs. The lack of difference in the susceptibility pattern indicates that clinically significant emergence of resistance does not occur in those patients who receive prolonged antifungal therapy.     

Effect of neutropenia and treatment delay on the response to antifungal agents in experimental disseminated candidiasis

Disseminated candidiasis is associated with a high rate of morbidity and mortality. The presence of neutrophils and the timely administration of antifungal agents are likely to be critical factors for a favorable therapeutic outcome of this syndrome. The effect of neutropenia on the temporal profile of the burden of Candida albicans in untreated mice and those treated with amphotericin B was determined using a pharmacodynamic model of disseminated candidiasis. A mathematical model was developed to describe the rate and extent of the C. albicans killing attributable to neutrophils and to amphotericin B. The consequences of a delay in the administration of amphotericin B, flucytosine, or micafungin were studied by defining dose-response relationships. Neutrophils caused a logarithmic decline in fungal burden in treated and untreated mice. The combination of amphotericin B and neutrophils resulted in a high rate of Candida killing and a sustained anti-C. albicans effect. In neutropenic mice, 5 mg/kg of body weight of amphotericin B was required to prevent progressive logarithmic growth. An increased delay in drug administration resulted in a reduction in the maximum effect to a point at which no drug effect could be observed. Neutrophils and the timely initiation of antifungal agents are critical determinants in the treatment of experimental disseminated candidiasis.     

Penetration of Candida biofilms by antifungal agents

A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing Candida. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. The rates of diffusion of either drug through biofilms of three strains of Candida albicans were similar. However, the rates of drug diffusion through biofilms of C. glabrata or C. krusei were faster than those through biofilms of C. parapsilosis or C. tropicalis. In all cases, after 3 to 6 h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms. The abilities of flucytosine, fluconazole, amphotericin B, and voriconazole to penetrate mixed-species biofilms containing C. albicans and Staphylococcus epidermidis (a slime-producing wild-type strain, RP62A, and a slime-negative mutant, M7) were also investigated. All four antifungal agents diffused very slowly through these mixed-species biofilms. In most cases, diffusion was slower with biofilms containing S. epidermidis RP62A, but amphotericin B penetrated biofilms containing the M7 mutant more slowly. However, the drug concentrations reaching the distal edges of the biofilms always substantially exceeded the MIC. Thus, although the presence of bacteria and bacterial matrix material undoubtedly retarded the diffusion of the antifungal agents, poor penetration does not account for the drug resistance of Candida biofilm cells, even in these mixed-species biofilms.     

In vitro and in vivo antifungal activities of DU-6859a, a fluoroquinolone, in combination with amphotericin B and fluconazole against pathogenic fungi.

DU-6859a is an investigational fluoroquinolone agent with potent bactericidal activity, but by itself it has no antifungal activity. When combined with amphotericin B (AmB), however, DU-6859a clearly enhanced the in vitro antifungal activity of AmB against Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, and Cryptococcus neoformans in microdilution checkerboard studies. Positive interactions of DU-6859a with AmB against Aspergillus fumigatus were dependent on the medium used; yeast nitrogen base supplemented with amino acids, ammonium sulfate, and 1% glucose was better for demonstrating synergism, while in RPMI 1640 medium, unexpected antagonism between the drugs occurred against three of the strains tested. In combination with fluconazole (Flu), DU-6859a increased the activity of Flu against C. albicans both in synthetic amino acid medium fungal and in supplemented yeast nitrogen base. An in vitro time-kill study revealed that DU-6859a combined with AmB significantly suppressed the regrowth of C. albicans compared with the suppression brought about by AmB used alone in a concentration-dependent fashion. Furthermore, in a model of C. albicans infection in mice, the fungal load in infected kidneys was significantly less in mice given the combination treatment of DU-6859a plus either AmB or Flu, and thus, the combination treatment resulted in prolonged survival of infected mice compared with treatment with either antifungal alone. The prolonged survival in mice given the combined treatment was also observed in mice with A. fumigatus infection, indicating that DU-6859a potentiated the actions of the antifungal agents in vivo as well as in vitro.     

临床分离真菌耐药性分析

目的监测分析临床分离真菌对抗菌药物的耐药现状,以加强抗真菌药物的合理应用。方法对我院2007年1月至2007年6月分离鉴定出的479株真菌用目前常用的5种抗真菌药物进行药物敏感性试验和分析。结果479株真菌中念珠菌和其他真菌分别占98.3%和1.7%,其中前3位依次是白念珠菌（69.5%）、光滑念珠菌（16.9%）和热带念珠菌（8.8%）。在5种抗真菌药物中,耐药率由高到低依次为伊曲康唑（12.7%）、氟康唑（9.8%）、伏立康唑（7.5%）、氟胞嘧啶（1.8%）和两性霉素B（1.0%）。结论临床分离的真菌对当前常用抗真菌药物的耐药菌株增多,应引起重视。     

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.     

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.     

Antifungal agents used for deep-seated mycotic infections

The main antifungal agents used for deep-seated mycotic infections are the broad-spectrum antifungal drug amphotericin B, the narrow-spectrum agent flucytosine, and the newer broad-spectrum agents miconazole and ketoconazole. Amphotericin B remains the cornerstone of antifungal therapy. For the treatment of cryptococcal meningitis, the current recommendation is for the combined use of amphotericin B and flucytosine. 2-Hydroxystilbamidine is used only in indolent cases of blastomycosis; however, this condition is usually treated with amphotericin B. Clinical experience with the newer agents is limited. Not all patients from whom fungal agents have been isolated require treatment; the extent of the fungal infection should be determined, when possible, for evaluation of the need for treatment.     

Collaborative investigation of variables in susceptibility testing of yeasts.

A multicenter study was performed to evaluate the effect of medium, incubation time (24 and 48 h), and temperature (30 and 35 degrees C) on intra- and interlaboratory variations in MICs of flucytosine, amphotericin B, and ketoconazole for yeasts. Testing was performed on coded isolates of Candida species (11 strains) and Cryptococcus neoformans (2 strains) by using a standard macrodilution protocol 11 laboratories. Four chemically defined media buffered to pH 7.0 with morpholinepropanesulfonic acid were evaluated, including buffered yeast nitrogen base, synthetic amino acid medium-fungal, RPMI 1640 medium, and high-resolution antifungal assay medium. Intralaboratory variability was less than or equal to fourfold for 97% of the replicate sets of data. The highest level of interlaboratory agreement, irrespective of antifungal agent or incubation conditions, was observed with RPMI 1640 medium. Intralaboratory variability was less than or equal to fourfold for 93% of the determinations with ketoconazole and 100% with flucytosine tested in RPMI 1640 medium at 35 degrees C for 24 h. Variability in amphotericin B results was less than or equal to fourfold for 81% of the determinations in RPMI 1640 medium at 35 degrees C for 48 h. The rank order of MICs within each antifungal test group was similar among the various laboratories and was generally in agreement with the reference rank order regardless of the test medium that we used.     

Synergism between the antifungal agents amphotericin B and alkyl glycerol ethers.

The alkyl glycerol ether rac-1-O-dodecylglycerol inhibited the growth of members of two genera of yeasts, Candida and Cryptococcus, and was strongly synergistic with amphotericin B. At one-half its MIC, dodecylglycerol decreased the MIC of amphotericin B by as much as 80-fold. This high degree of synergism between dodecylglycerol and amphotericin B was demonstrated against a number of species of yeasts including Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii. All fractional inhibitory concentrations (for all strains and species) were calculated to be less than 1, and most were less than 0.6, again demonstrating strong synergism. Other alkyl glycerol ethers with alkyl chain lengths ranging from 8 to 18 carbon atoms were also found to be synergistic with amphotericin B against C. neoformans and C. albicans. Electron microscopy experiments showed that C. neoformans grown in the presence of dodecylglycerol had severely abnormal, deformed capsules. Although the mechanism of action of dodecylglycerol is not known, dodecylglycerol was not simply acting as a detergent. The natural detergent sodium deoxycholate could not substitute for dodecylglycerol. At comparable and higher concentrations, sodium deoxycholate had no fungicidal effect on its own, nor did it potentiate the activity of amphotericin B. Dodecylglycerol did not interact synergistically with the water-soluble antifungal agent fluconazole. The lipid-soluble hydrophobic properties of amphotericin B appear to be important for this synergistic effect, in that alkyl glycerol ethers could promote synergism with amphotericin B by potentially increasing the interaction between membrane-bound ergosterol and amphotericin B.     

Antifungal agents used for deep-seated mycotic infections

The main antifungal agents used for deep-seated mycotic infections are the broad-spectrum antifungal drug amphotericin B, the narrow-spectrum agent flucytosine, and the newer broad-spectrum agents ketoconazole, miconazole, and itraconazole. Amphotericin B remains the cornerstone of antifungal therapy. For the treatment of cryptococcal meningitis, the current recommendation is for the combined use of amphotericin B and flucytosine. Published clinical experience with the newer agents is limited. Not all patients from whom fungal agents have been isolated require treatment; the extent of the fungal infection should be determined, when possible, for evaluation of the need for treatment.     

Comparison of a spectrophotometric microdilution method with RPMI-2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testing of amphotericin B, flucytosine, and fluconazole against Candida albicans.

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by visual and subjective inspection of growth inhibition after 48 h of incubation. An alternative microdilution procedure was compared with the M27-P method for determination of the amphotericin B, flucytosine, and fluconazole susceptibilities of 8 American Type Culture Collection strains (6 of them were quality control or reference strains) and 50 clinical isolates of candida albicans. This microdilution method uses as culture medium RPMI 1640 supplemented with 18 g of glucose per liter (RPMI-2% glucose). Preparation of drugs, basal medium, and inocula was done by following the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h. Increased growth of C. albicans in RPMI-2% glucose and its spectrophotometric reading allowed for the rapid (24 h) and objective calculation of MIC endpoints compared with previous microdilution methods with standard RPMI 1640. Nevertheless, good agreement was shown between the M27-P method and this microdilution test. The MICs obtained for the quality control or reference strains by the microdilution method were in the ranges published for those strains. For clinical isolates, the percentages of agreement were 100% for amphotericin B and fluconazole and 98.1% for flucytosine. These data suggest that this microdilution method may serve as a less subjective and more rapid alternative to the M27-P method for antifungal susceptibility testing of yeasts.     

Effects of amphotericin B and fluconazole on the extracellular and intracellular growth of Candida albicans.

The effects of amphotericin B and fluconazole on the extracellular and intracellular growth of Candida albicans were studied. With respect to the extracellular growth of C. albicans, antifungal activity was measured in terms of MICs and minimal fungicidal concentrations as well as by determination of the concentration that effectively killed (greater than 99.9%) C. albicans in the absence or presence (amphotericin B only) of serum. Amphotericin B was highly active in terms of killing, even at an increased inoculum size. In the presence of serum, amphotericin B activity was substantially reduced. For fluconazole, activity was restricted to inhibition of fungal growth, even after the inoculum size was reduced. With respect to the intracellular growth of C. albicans, antifungal activity was measured by using monolayers of murine peritoneal macrophages infected with C. albicans and was measured in terms of inhibition of germ tube formation as well as effective killing (greater than 99%) of C. albicans. Amphotericin B was highly active against C. albicans. At an increased ratio of infection, amphotericin B activity was slightly reduced. Fluconazole had no antifungal activity. Neither a reduction in the ratio of infection nor exposure of C. albicans to fluconazole prior to macrophage ingestion resulted in activity against intracellular C. albicans by fluconazole. Previous exposure of C. albicans to amphotericin B resulted in increased intracellular activity of amphotericin B. The intracellular antifungal activity of the combination of fluconazole with amphotericin B was less than that of amphotericin B alone. Amphotericin B showed fungicidal activity against C. albicans growing both extracellularly and intracellularly, whereas fluconazole inhibited growth only of extracellular C. albicans. A slight antagonistic effect between fluconazole and amphotericin B was found with respect to intracellular as well as extracellular C. albicans.     

In vitro activity of A-192411.29, a novel antifungal lipopeptide

A-192411.29 is a novel antifungal agent derived from the structural template of the natural product echinocandin. The in vitro activity of A-192411.29 against common pathogenic yeasts was assessed by National Committee for Clinical Laboratory Standards method M27-A. It demonstrated broad-spectrum, fungicidal activity and was active against the most clinically relevant yeasts, such as Candida albicans, Candida tropicalis, and Candida glabrata, as well as less commonly encountered Candida species; in general, its potency on a weight basis was comparable to that of amphotericin B. It maintained potent in vitro activity against Candida strains with reduced susceptibilities to fluconazole and amphotericin B. The in vitro activity of A-192411.29 against Cryptococcus neoformans was comparable to its activity against Candida spp. However, A-192411.29 did not demonstrate complete growth inhibition of Aspergillus fumigatus by the broth microdilution method used. A-192411.29 possesses an antifungal profile comparable to or better than those of fluconazole and amphotericin B against pathogenic yeasts, including strains resistant to fluconazole or amphotericin B, suggesting that it may be a therapeutically useful new antifungal drug.     

Results obtained with various antifungal susceptibility testing methods do not predict early clinical outcome in patients with cryptococcosis

The in vitro susceptibilities of Cryptococcus neoformans isolates from consecutive human immunodeficiency virus-positive and -negative patients to the antifungal agents fluconazole, amphotericin B, and flucytosine were determined by different techniques, including the CLSI method, Etest, and broth microdilution in yeast nitrogen base (YNB) medium, during a multicenter prospective study in France. The relationship between the in vitro data and the clinical outcome 2 weeks after the initiation of antifungal therapy was assessed. In addition, the correlation between the strain serotype and the in vitro activities of the antifungals was determined, and the susceptibility results obtained with the different techniques were also compared. Thirty-seven patients received a combination of amphotericin B with flucytosine as first-line therapy, 22 were treated with amphotericin B alone, and 15 received fluconazole alone. Whatever the antifungal tested, there was no trend toward higher MICs for strains isolated from patients who failed to respond to a given therapy compared to those from patients who did not with either the CLSI method, Etest, or broth microdilution in YNB medium. The MICs obtained by the CLSI or Etest method were significantly lower for serotype D strains than for serotype A strains for both fluconazole and amphotericin B, while flucytosine MICs were not different according to serotype. These findings suggest that the in vitro antifungal susceptibility of C. neoformans, as determined with the techniques used, is not able to predict the early clinical outcome in patients with cryptococcosis.