Sevtap Arikan

In Vitro Synergy of Caspofungin and Amphotericin B against Aspergillus and Fusarium spp.

ABSTRACT We investigated the in vitro interaction of caspofungin and amphotericin B for clinical isolates of Aspergillus and Fusarium. Synergy tests were performed using the checkerboard method and following the NCCLS M38-P guidelines in Antibiotic Medium 3 broth supplemented to 2% glucose. Antagonism was not observed for any of the isolates tested. Caspofungin and amphotericin B were synergistic or synergistic to additive for at least half of the isolates.

In Vitro Susceptibility Testing Methods for Caspofungin against Aspergillus and Fusarium Isolates

ABSTRACT We investigated the relevance of prominent reduction in turbidity macroscopically (MIC) and formation of aberrant hyphal tips microscopically (minimum effective concentration; MEC) in measuring the in vitro activity of caspofungin against Aspergillus andFusarium. Caspofungin generated low MICs and MECs againstAspergillus, but not for Fusarium. While MICs increased inconsistently when the incubation time was prolonged, MEC appeared as a stable and potentially relevant endpoint in testing in vitro caspofungin activity.

Current status of antifungal susceptibility testing methods

Antifungal susceptibility testing is a very dynamic field of medical mycology. Standardization of in vitro susceptibility tests by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST), and current availability of reference methods constituted the major remarkable steps in the field. Based on the established minimum inhibitory concentration (MIC) breakpoints, it is now possible to determine the susceptibilities of Candida strains to fluconazole, itraconazole, voriconazole, and flucytosine. Moreover, utility of fluconazole antifungal susceptibility tests as an adjunct in optimizing treatment of candidiasis has now been validated. While the MIC breakpoints and clinical significance of susceptibility testing for the remaining fungi and antifungal drugs remain yet unclear, modifications of the available methods as well as other methodologies are being intensively studied to overcome the present drawbacks and limitations. Among the other methods under investigation are Etest, colorimetric microdilution, agar dilution, determination of fungicidal activity, flow cytometry, and ergosterol quantitation. Etest offers the advantage of practical application and favorable agreement rates with the reference methods that are frequently above acceptable limits. However, MIC breakpoints for Etest remain to be evaluated and established. Development of commercially available, standardized colorimetric panels that are based on CLSI method parameters has added more to the antifungal susceptibility testing armamentarium. Flow cytometry, on the other hand, appears to offer rapid susceptibility testing but requires specified equipment and further evaluation for reproducibility and standardization. Ergosterol quantitation is another novel approach, which appears potentially beneficial particularly in discrimination of azole-resistant isolates from heavy trailers. The method is yet investigational and requires to be further studied. Developments in methodology and applications of antifungal susceptibility testing will hopefully provide enhanced utility in clinical guidance of antifungal therapy. However, and particularly in immunosuppressed host, in vitro susceptibility is and will remain only one of several factors that influence clinical outcome.

Comparative in vitro activities of posaconazole, voriconazole, itraconazole, and amphotericin B against Aspergillus and Rhizopus, and synergy testing for Rhizopus

We compared the in vitro activities of posaconazole, voriconazole, itraconazole, and amphotericin B against clinical isolates of Aspergillus spp. and Rhizopus spp., and explored the in vitro interaction between posaconazole and amphotericin B against Rhizopus spp. Clinical strains of 82 Aspergillus spp. (43 Aspergillus fumigatus, 29 A. flavus, 7 A. niger, 2 A. terreus, 1 A. nidulans) and 11 Rhizopus oryzae isolates were tested in accordance with CLSI M38-A microdilution guidelines. In vitro activity of posaconazole against Aspergillus spp. was also investigated with the Etest. The combination of posaconazole and amphotericin B against R. oryzae isolates was investigated by the checkerboard methodology. Voriconazole was the most active drug in vitro against Aspergillus spp., followed by posaconazole, itraconazole, and amphotericin B, in order of decreasing activity. In studies with R. oryzae isolates, posaconazole was found to be the most potent drug followed by itraconazole and amphotericin B. Voriconazole had no meaningful activity against Rhizopus. Posaconazole Etest MICs (microg/ml) with Aspergillus spp. were found to be considerably lower than those obtained with the CLSI microdilution method (4-9 and 3-7 two-fold lower than CLSI MICs at 24 and 48 h, respectively). The interaction between posaconazole and amphotericin B was indifferent for all R. oryzae isolates tested; importantly no antagonism was observed.

Comparative Evaluation of Disk Diffusion with Microdilution Assay in Susceptibility Testing of Caspofungin against Aspergillus and Fusarium Isolates

ABSTRACT We compared the disk diffusion and broth microdilution methods for susceptibility testing of caspofungin against Aspergillus (n = 78) and Fusarium (n = 22) isolates. Microdilution testing followed the NCCLS M-38P guidelines but was performed in antibiotic medium 3 supplemented to 2% glucose (AM3). Disk diffusion assays were performed on AM3 agar plates with a 2-μg caspofungin disk. By both methods, caspofungin showed favorable activity against Aspergillus isolates and no activity against Fusarium isolates. In the disk-based format, intrazonal growth that was not influenced by the drug concentration gradient was consistently observed for all of the Aspergillus isolates tested.

Susceptibility testing of voriconazole, fluconazole, itraconazole and amphotericin B against yeast isolates in a Turkish University Hospital and effect of time of reading

Voriconazole is a promising azole effective against a variety of fungi, including yeasts. In this study, we tested in vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against some ATCC and reference strains and 250 clinical yeast isolates. We also evaluated the effect of time of reading on MIC results. Voriconazole was the most active agent against Candida and Trichosporon isolates, including the putatively fluconazole-resistant C. krusei (MIC(90) 0.25 microg/ml) and C. glabrata (MIC(90) 0.5 microg/ml). Amphotericin B MICs were scattered in a considerably narrow range in both RPMI 1640 and Antibiotic Medium 3. MICs at 24 hours and 48 hours were similar in general for all antifungals tested. The highest percentage of strains that showed 24-hour and 48-hour MICs within +/-1-log(2) dilution was observed for amphotericin B tested in RPMI (99%), and the lowest for amphotericin B tested in Antibiotic Medium 3 (80%). In conclusion, voriconazole is very effective against a wide spectrum of Candida species and 24-hour readings could substitute 48-hour MIC evaluation.

Lipid-based antifungal agents : Current status

Immunocompromised patients are well known to be predisposed to developing invasive fungal infections. These infections are usually difficult to diagnose and more importantly, the resulting mortality rate is high. The limited number of antifungal agents available and their high rate of toxicity are the major factors complicating the issue. However, the development of lipid-based formulations of existing antifungal agents has opened a new era in antifungal therapy. The best examples are the lipid-based amphotericin B preparations, amphotericin B lipid complex (ABLC; Abelcet), amphotericin B colloidal dispersion (ABCD; Amphotec or Amphocil), and liposomal amphotericin B (AmBisome). These formulations have shown that antifungal activity is maintained while toxicity is reduced. This progress is followed by the incorporation of nystatin into liposomes. Liposomal nystatin formulation is under development and studies of it have provided encouraging data. Finally, lipid-based formulations of hamycin, miconazole, and ketoconazole have been developed but remain experimental. Advances in technology of liposomes and other lipid formulations have provided promising new tools for management of fungal infections.

Comparison of two methods and three end points in determination of in vitro activity of micafungin against Aspergillus spp.

ABSTRACT We investigated the in vitro activity of micafungin against clinical Aspergillus isolates (n = 37) (Aspergillusfumigatus [n = 21], Aspergillusflavus [n = 14], and Aspergillus niger [n = 2]) by using NCCLS M38A microdilution and an investigational disk diffusion assay. Microdilution assay results were evaluated by using the end points of a MIC-2 (measured in micrograms per milliliter) and minimum effective concentration (MEC, measured in micrograms per milliliter; the lowest concentration of micafungin that produces short and aberrant hyphal branchings microscopically). Disk diffusion results were interpreted by measuring the zone(s) of inhibition (ZOI, measured in millimeters). Micafungin proved to be similarly active against all Aspergillus species tested. At 24 h, MIC-2s and MECs were identical. At 48 h, however, MIC-2s increased unpredictably, leading to the loss of a consistent correlation between the two end points. MECs and ZOI remained consistent and correlated at both reading times, suggesting their use as relevant end points in susceptibility testing of micafungin against Aspergillus. All Aspergillus isolates yielded intrazonal growth on disk diffusion agar plates. The intrazonal colonies contained short, aberrant hyphal branchings microscopically. The in vivo significance of these findings remains to be further investigated.

In Vitro Activity of Nystatin Compared with Those of Liposomal Nystatin, Amphotericin B, and Fluconazole against Clinical Candida Isolates

ABSTRACT We investigated the in vitro activity of nystatin and liposomal nystatin against 103 Candida isolates to determine the effect of both time and medium on MICs. We also compared the nystatin MICs with those of amphotericin B and fluconazole. Testing was performed in accordance with the National Committee for Clinical Laboratory Standards M27-A microdilution methodology with RPMI 1640, RPMI 1640 supplemented with glucose to 2% (RPMI-2), and antibiotic medium 3 supplemented with glucose to 2% (AM3). While nystatin MICs were similar to or slightly lower than liposomal nystatin MICs in RPMI 1640 and RPMI-2, they were markedly higher than liposomal nystatin MICs in AM3. Use of AM3 and determination of the MIC after 24 h of incubation provided a slightly wider range of liposomal nystatin MICs (0.06 to >16 μg/ml). Under these conditions, the MICs at which 90% of isolates were inhibited of nystatin and liposomal nystatin were 2 and 1 μg/ml, respectively. Nystatin and liposomal nystatin in general showed good activity against all Candida spp. tested. Although the MICs of nystatin and liposomal nystatin tended to rise in parallel with the amphotericin B MICs, nystatin and liposomal nystatin MICs of 1 to 2 and 0.5 to 1 μg/ml, respectively, were obtained for seven and six, respectively, of nine isolates for which amphotericin B MICs were ≥0.25 μg/ml. No correlation between fluconazole and nystatin or liposomal nystatin MICs was observed. As amphotericin B MICs of ≥0.25 μg/ml correlate with in vitro resistance, these results suggest that liposomal nystatin might have activity against some amphotericin B-resistant isolates. In vivo testing in animal models is required for clarification of this issue.

Comparison of NCCLS microdilution method and Etest in antifungal susceptibility testing of clinical Trichosporon asahii isolates

We investigated the in vitro activity of amphotericin B, fluconazole, and itraconazole against clinical Trichosporon asahii isolates (n = 43) by NCCLS M27A reference microdilution method and explored the correlation between Etest and NCCLS reference method. Microdilution MIC ranges following 48 h of incubation were 1-8, 0.25-16, and 0.06-4 microg/ml for amphotericin B, fluconazole, and itraconazole, respectively. The corresponding Etest MIC ranges were determined as 0.125- > 8, 0.25- > 64, and 0.03-8 microg/ml. Of interest, Etest tended to produce lower amphotericin B MICs and widen the MIC range compared to microdilution. The influence of Etest on fluconazole and itraconazole MICs was in contrary with that observed for amphotericin B. Etest MICs of fluconazole and itraconazole tended to be higher than microdilution MICs. The wider range of amphotericin B MICs obtained by using Etest methodology may facilitate discrimination of isolates with reduced susceptibility to amphotericin B. However, clinical significance of these findings remain yet unknown and determination of MIC breakpoint values is required.