In-vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi

The in-vitro fungistatic and fungicidal activities of voriconazole were compared with those of itraconazole and amphotericin B. MICs for 110 isolates belonging to 11 species of filamentous fungi were determined by a broth microdilution adaptation of the method recommended by the National Committee for Clinical Laboratory Standards. Minimum lethal concentrations (MLCs) of the three antifungal agents were also determined. The MIC ranges of the three compounds were comparable for Aspergillus flavus, Aspergillus fumigatus, Cladophialophora bantiana and Exophiala dermatitidis. Voriconazole and itraconazole were more active than amphotericin B against Fonsecaea pedrosoi, but the two azole agents were less active against Sporothrix schenckii. Voriconazole was more active than itraconazole or amphotericin B against Scedosporium apiospermum, but less active than the other two agents against two mucoraceous moulds, Absidia corymbifera and Rhizopus arrhizus. Voriconazole and amphotericin B were more active than itraconazole against Fusarium solani. With the exception of S. apiospermum, all the moulds tested had MLC50 values of < or =2 mg/L and MLC90 values of < or =4 mg/L against amphotericin B. Voriconazole and itraconazole showed fungicidal effects against five of the 1 1 moulds tested (A. flavus, A. fumigatus, C. bantiana, E. dermatitidis and F. pedrosoi) with MLC90 values of < or =2 mg/L. In addition, voriconazole was fungicidal for Phialophora parasitica. Our results suggest that voriconazole could be effective against a wide range of mould infections in humans.     

In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts

The in vitro activity of the novel triazole antifungal agent posaconazole (Noxafil; SCH 56592) was assessed in 45 laboratories against approximately 19,000 clinically important strains of yeasts and molds. The activity of posaconazole was compared with those of itraconazole, fluconazole, voriconazole, and amphotericin B against subsets of the isolates. Strains were tested utilizing Clinical and Laboratory Standards Institute broth microdilution methods using RPMI 1640 medium (except for amphotericin B, which was frequently tested in antibiotic medium 3). MICs were determined at the recommended endpoints and time intervals. Against all fungi in the database (22,850 MICs), the MIC(50) and MIC(90) values for posaconazole were 0.063 microg/ml and 1 mug/ml, respectively. MIC(90) values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 mug/ml. In comparative studies against subsets of the isolates, posaconazole was more active than, or within 1 dilution of, the comparator drugs itraconazole, fluconazole, voriconazole, and amphotericin B against approximately 7,000 isolates of Candida and Cryptococcus spp. Against all molds (1,702 MICs, including 1,423 MICs for Aspergillus isolates), posaconazole was more active than or equal to the comparator drugs in almost every category. Posaconazole was active against isolates of Candida and Aspergillus spp. that exhibit resistance to fluconazole, voriconazole, and amphotericin B and was much more active than the other triazoles against zygomycetes. Posaconazole exhibited potent antifungal activity against a wide variety of clinically important fungal pathogens and was frequently more active than other azoles and amphotericin B.     

In-vitro susceptibility of Aspergillus spp. isolates to amphotericin B and itraconazole

The MICs of amphotericin B and itraconazole for 230 isolates of Aspergillus spp., comprising 156 Aspergillus fumigatus, 20 Aspergillus terreus, 22 Aspergillus flavus, 17 Aspergillus nidulans and 15 Aspergillus niger, were determined by a broth microdilution method with RPMI 1640 medium. No isolate was detected with an MIC of amphotericin B >2 mg/L. Itraconazole MICs >16 mg/L were detected for four Aspergillus fumigatus and one Aspergillus nidulans isolates.     

Germinated and nongerminated conidial suspensions for testing of susceptibilities of Aspergillus spp. to amphotericin B, itraconazole, posaconazole, ravuconazole, and voriconazole

The effect of germinated and nongerminated conidia of Aspergillus spp. on the fungistatic (National Committee for Clinical Laboratory Standards document M38-P) and fungicidal activities (MICs and minimal fungicidal concentrations [MFCs] respectively) of amphotericin B, itraconazole, posaconazole (SCH56592), ravuconazole (BMS-207147), and voriconazole was evaluated. MFCs were the lowest drug dilutions that showed fewer than three colonies (99.9% killing). Overall, the MICs (0.12 to 4 microg/ml) and MFCs (0.5 to >8 microg/ml) of all of the agents tested with both inocula were the same or within 2 dilutions for the 72 isolates. Therefore, MICs and MFCs can be obtained with convenient and standardized nongerminated conidia.     

Treatment failure in invasive aspergillosis: susceptibility of deep tissue isolates following treatment with amphotericin B

OBJECTIVES: To determine whether treatment failure in invasive aspergillosis (IA) is the result of resistance of Aspergillus spp. isolates to amphotericin B. METHODS: Six Aspergillus fumigatus and six Aspergillus flavus isolates cultured from deep tissue biopsies in 11 patients with haematological malignancies during 1991-1998 were tested. A method based on the NCCLS M38-A broth microdilution method, with colorimetric determination of MICs, was used to determine the MICs of amphotericin B and itraconazole. RESULTS: All A. fumigatus isolates were susceptible to amphotericin B (MIC 0.25-0.5 mg/L), as were three A. flavus isolates (MIC 1 mg/L), but three were less susceptible (MIC 2 mg/L). All isolates were susceptible to itraconazole (MIC 0.125-0.25 mg/L). All patients had been treated with amphotericin B, having received a median of 12 days of treatment when the tissue was obtained. CONCLUSION: The difficulty in treating IA may not be because of the susceptibility of the isolates, but because of poor penetration of antifungal agents into infected tissue. Aspergillus spp. invade blood vessels causing thrombosis and tissue infarction, and therefore it may be difficult for antifungal drugs to exceed MICs in infected tissues. This highlights the need for different treatment strategies, such as surgery and the administration of cytokines.     

In vitro interaction of terbinafine with itraconazole,fluconazole, amphotericin B and 5-flucytosine against Aspergillus spp

We investigated the in vitro interaction of terbinafine with itraconazole, fluconazole, amphotericin B and 5-flucytosine, against Aspergillus spp. We tested three isolates of Aspergillus fumigatus (one resistant to itraconazole), and two each of Aspergillus flavus, Aspergillus niger and Aspergillus terreus. We employed a broth microdilution-based method derived from an in vivo validated method capable of detecting itraconazole resistance in A. fumigatus. We studied the effect on the MICs by calculation of the fractional inhibitory concentration (FIC) and fractional fungicidal concentration (FFC) (99.99% kill). Itraconazole and terbinafine were synergic or additive in all strains (FIC = 0.15-1.0). Fluconazole and terbinafine were synergic with A. fumigatus, A. terreus and A. flavus (FIC = 0.3-0.5) and indifferent with A. niger (FIC = 2) isolates. Amphotericin B and terbinafine were mostly indifferent or antagonistic (FIC = 1.0-4.02). Flucytosine and terbinafine were usually indifferent or antagonistic (FIC = 0.63-8.5). FFCs were generally in accord with FICs. The use of terbinafine in combination therapy for Aspergillus infections with azoles seems promising, whereas terbinafine and amphotericin B or flucytosine in combination were less effective.     

Differential activity of triazoles in two-drug combinations with the echinocandin caspofungin against Aspergillus fumigatus

OBJECTIVES: We investigated the in vitro activity of various triazoles in two-drug combinations with the echinocandin caspofungin against clinical isolates of Aspergillus fumigatus.Method: Conidial suspensions were prepared from 20 clinical isolates of A. fumigatus highly susceptible to itraconazole, voriconazole, posaconazole and ravuconazole (MIC-0 range 0.125-1 mg/L), and caspofungin (MIC-0 range 32-64 mg/L). The in vitro susceptibility of A. fumigatus to two-drug combinations of itraconazole, voriconazole, posaconazole and ravuconazole with caspofungin was evaluated by the fractional inhibitory concentration index (FICI) method. RESULTS: Two-drug combinations of caspofungin with itraconazole (FICI = 0.49 +/- 0.04) or posaconazole (FICI = 0.32 +/- 0.09) provided synergic interaction. On the other hand, ravuconazole (FICI = 0.61 +/- 0.31) and voriconazole (FICI = 1.61 +/- 0.42) in combination with caspofungin showed no interaction against A. fumigatus. CONCLUSIONS: Our data show that the in vitro antifungal efficacies of combinations of members from two different classes are not always similar and hence are not predictable.     

Flow cytometry antifungal susceptibility testing of Aspergillus fumigatus and comparison of mode of action of voriconazole vis-à-vis amphotericin B and itraconazole

Aspergillus fumigatus isolates were tested with three antifungals by flow cytometry (FC) and fluorescence-activated cell sorting. FC results after 4 h correlated well with MICs obtained by the NCCLS M38-A method; voriconazole exhibited fungicidal activity, albeit to a lesser extent than amphotericin B, but to a greater extent than itraconazole.     

Differential fungicidal activities of amphotericin B and voriconazole against Aspergillus species determined by microbroth methodology

Antifungal agents may differ in their fungicidal activities against Aspergillus spp. In order to compare the fungicidal activities of voriconazole and amphotericin B against 40 isolates of Aspergillus fumigatus, A. flavus, and A. terreus, we developed a new microbroth colorimetric method for assessing fungicidal activities and determining minimal fungicidal concentrations (MFCs). This methodology follows the antifungal susceptibility testing reference method M-38A for MIC determination. After drug removal and addition of fresh medium, growth of viable conidia adhering to the bottoms of the microtitration wells was assessed by a colorimetric assay of metabolic activity after 24 h of incubation. The new method was faster (six times), reproducible (92 to 97%), and in agreement with culture-based MFCs (91 to 100%). Differential fungicidal activities of voriconazole and amphotericin B were found among the three Aspergillus species, with A. fumigatus and A. flavus having the lowest (1 and 2 mg/liter, respectively) and A. terreus the highest (>16 mg/liter) median amphotericin B MFCs; A. flavus had a lower median voriconazole MFC (4 mg/liter) than the other species (>8 mg/liter; P < 0.05). Amphotericin B was fungicidal (MFC/MIC 4) against 94% of A. fumigatus and 84% of A. terreus isolates. The new methodology revealed a concentration-dependent sigmoid pattern of fungicidal effects, indicating that fungicidal activity is not an all-or-nothing phenomenon and that some degree of fungicidal action can be found even for agents considered fungistatic based on the MFC/MIC ratio.     

In vitro activity of a new triazole BAL4815, the active component of BAL8557 (the water-soluble prodrug), against Aspergillus spp

OBJECTIVES: BAL4815 is the active component of the antifungal triazole agent BAL8557 (the water-soluble prodrug). We compared the in vitro activity of BAL4815 with that of itraconazole, voriconazole, caspofungin and amphotericin B against 118 isolates of Aspergillus comprising four different species (fumigatus, terreus, flavus and niger); the isolates were pre-selected to include 16 isolates demonstrating in vitro resistance to other agents. METHODS: Susceptibilities were determined for BAL4815, amphotericin B, itraconazole and voriconazole using the microdilution plate modification of the NCCLS M38-A method with RPMI 1640 buffered to pH 7.0 with MOPS; for caspofungin the method was modified using incubation in a gas mixture of 1% O2/5% CO2/94% N2 to aid reading. MFCs (> or =99% kill) were also determined for all drugs other than caspofungin. RESULTS: For all isolates, geometric mean (GM) MIC values and ranges (in mg/L) were: BAL4815, 0.620 and 0.125-2.0; itraconazole, 0.399 and 0.063->8.0; voriconazole, 0.347 and 0.125-8.0; caspofungin, 0.341 and 0.125-4.0; amphotericin B, 0.452 and 0.06-4.0. No significant differences in susceptibility to BAL4815 were seen between species and in contrast to itraconazole no isolates demonstrated MICs >2.0 mg/L. For all isolates, GM MFC values and ranges (in mg/L) were: BAL4815, 1.68 and 0.25->8.0; itraconazole, 1.78 and 0.06->8.0; voriconazole, 1.09 and 0.25->8.0; amphotericin B, 0.98 and 0.25->4.0. CONCLUSIONS: BAL4815 demonstrated promising antifungal activity against all four Aspergillus species in vitro including strains resistant to itraconazole, caspofungin or amphotericin B.     

Correlation between the E test and the CLSI M-38 A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus

The in vitro activities of amphotericin B, itraconazole, and voriconazole against 283 clinical isolates of Aspergillus fumigatus were studied by comparing the E test method with the reference procedure NCCLS (CLSI) M-38A. The methods were considered to agree when the results of the MICs by E test were within +/-2 dilutions of the MICs obtained by CLSI M-38 A. Agreement of readings at 24, 48, and 72 h of incubation was as follows: amphotericin B: 4.2%, 98%, and 40.6%; itraconazole: 92.5%, 100%, and 89.9%; and voriconazole: 98.9%, 100%, and 99.7%. The correlation between methods to classify the strains as "susceptible" or "resistant" was very good for itraconazole and voriconazole after 48 h of incubation, but 23% of the strains were incorrectly classified by E test for amphotericin B. The E test is an efficacious method for antifungal susceptibility testing in A. fumigatus for itraconazole and voriconazole when the plates are read after 48 h of incubation. The use of the E test to study the activity of amphotericin B should be avoided.     

Antifungal activities of posaconazole, ravuconazole, and voriconazole compared to those of itraconazole and amphotericin B against 239 clinical isolates of Aspergillus spp. and other filamentous fungi: report from SENTRY Antimicrobial Surveillance Program, 2000

Posaconazole, ravuconazole, and voriconazole are new triazole derivatives that possess potent, broad-spectrum antifungal activity. We evaluated the in vitro activity of these investigational triazoles compared with that of itraconazole and amphotericin B against 239 clinical isolates of filamentous fungi from the SENTRY Program, including Aspergillus spp. (198 isolates), Fusarium spp. (7 isolates), Penicillium spp. (19 isolates), Rhizopus spp. (4 isolates), Mucor spp. (2 isolates), and miscellaneous species (9 isolates). The isolates were obtained from 16 different medical centers in the United States and Canada between January and December 2000. In vitro susceptibility testing was performed using the microdilution broth method outlined in the National Committee for Clinical Laboratory Standards M38-P document. Overall, posaconazole was the most active compound, inhibiting 94% of isolates at a MIC of < or = 1 microg/ml, followed by voriconazole (91%), amphotericin B (89%), ravuconazole (88%), and itraconazole (70%). All three new triazoles demonstrated excellent activity (MIC, < or = 1 microg/ml) against Aspergillus spp. (114 Aspergillus fumigatus, 22 Aspergillus niger, 13 Aspergillus flavus, 9 Aspergillus versicolor, 8 Aspergillus terreus, and 32 Aspergillus spp.): posaconazole (98%), voriconazole (98%), ravuconazole (92%), amphotericin B (89%), and itraconazole (72%). None of the triazoles were active against Fusarium spp. (MIC at which 50% of the isolates tested were inhibited [MIC(50)], >8 microg/ml) or Mucor spp. (MIC(50), >8 microg/ml). Posaconazole and ravuconazole were more active than voriconazole against Rhizopus spp. (MIC(50), 1 to 2 microg/ml versus >8 microg/ml, respectively). Based on these results, all three new triazoles exhibited promising activity against Aspergillus spp. and other less commonly encountered isolates of filamentous fungi. The clinical value of these in vitro data remains to be seen, and in vitro-in vivo correlation is needed for both new and established antifungal agents. Surveillance efforts should be expanded in order to monitor the spectrum of filamentous fungal pathogens and their in vitro susceptibility as these new antifungal agents are introduced into clinical use.     

In vitro activities of three licensed antifungal agents against spanish clinical isolates of Aspergillus spp

The aim of the present study was to identify retrospectively trends in the species distributions and the susceptibility patterns of Aspergillus species causing fungal infections in Spanish medical centers from 2000 to 2002. The susceptibilities of 338 isolates to amphotericin B, itraconazole, and voriconazole were tested. Aspergillus fumigatus was the most common species (54.7%), followed by Aspergillus terreus (14.8%) and Aspergillus flavus (13.9%). Non-A. fumigatus species were encountered in 45.3% of the samples studied. The majority of Aspergillus isolates were obtained from respiratory tract specimens, followed by ear and skin samples. The geometric mean (GM) MIC of amphotericin B was 0.56 micro g/ml, and the amphotericin B MIC was >2 micro g/ml for 16 isolates (4.7%). Nine of them were A. terreus. The GM MIC of itraconazole was 0.37, and the itraconazole MIC was >4 micro g/ml for 12 (3.5%) isolates. The voriconazole MICs were also high for 8 of the 12 strains for which itraconazole MICs were high (voriconazole MIC range, 2 to 8 micro g/ml).     

Comparison of the Sensititre YeastOne colorimetric antifungal panel and Etest with the NCCLS M38-A method to determine the activity of amphotericin B and itraconazole against clinical isolates of Aspergillus spp

OBJECTIVE: The evaluation of the Sensititre YeastOne and Etest methods for determining susceptibility to amphotericin B and itraconazole by comparing the MICs obtained by these methods at different times of reading with those of the M38-A broth microdilution method. METHODS: Sixty-three clinical isolates of Aspergillus spp. (23 Aspergillus flavus, 24 Aspergillus fumigatus, nine Aspergillus niger, three Aspergillus glaucus, two Aspergillus terreus and two Aspergillus flavipes) were assayed. Two itraconazole-resistant strains (NCPF7100 and 7099) were also included. RESULTS: Itraconazole MICs for the two resistant strains were >4 mg/L by the three methods. The overall agreement (+/-2 log2) between M38-A (48 h) and colorimetric (48 h) method was 93.4% for amphotericin B and 90.2% for itraconazole. By the Etest, the best agreement with M38-A was obtained when readings were made at 24 h: 88.5% for amphotericin B and 67.2% for itraconazole. Etest MICs were higher for all species except A. niger. CONCLUSIONS: The colorimetric method appears to be a suitable alternative procedure for antifungal susceptibility testing of Aspergillus spp. and is able to detect resistance to itraconazole. The range of MICs for amphotericin B by Etest is wider and for some strains is >16 mg/L, suggesting that this method could be useful for detecting resistant strains as occur in yeasts.     

In vitro antifungal susceptibilities of Trichosporon species

The in vitro activities of amphotericin B, itraconazole, fluconazole, voriconazole, posaconazole, and ravuconazole against 39 isolates of Trichosporon spp. were determined by the NCCLS M27-A microdilution method. The azoles tested appeared to be more potent than amphotericin B. Low minimal fungicidal concentration/MIC ratios were observed for voriconazole, posaconazole, and ravuconazole, suggesting fungicidal activity.     

Antifungal susceptibilities of Aspergillus fumigatus clinical isolates obtained in Nagasaki, Japan

We investigated the triazole, amphotericin B, and micafungin susceptibilities of 196 A. fumigatus clinical isolates in Nagasaki, Japan. The percentages of non-wild-type (non-WT) isolates for which MICs of itraconazole, posaconazole, and voriconazole were above the ECV were 7.1%, 2.6%, and 4.1%, respectively. A G54 mutation in cyp51A was detected in 64.2% (9/14 isolates) and 100% (5/5 isolates) of non-WT isolates for itraconazole and posaconazole, respectively. Amphotericin B MICs of ≥2 μg/ml and micafungin minimum effective concentrations (MECs) of ≥16 μg/ml were recorded for two and one isolates, respectively.     

Isothermal microcalorimetry: a novel method for real-time determination of antifungal susceptibility of Aspergillus species

We evaluated microcalorimetry for real-time susceptibility testing of Aspergillus spp. based on growth-related heat production. The minimal heat inhibitory concentration (MHIC) for A. fumigatus ATCC 204305 was 1 mg/L for amphotericin B, 0.25 mg/L for voriconazole, 0.06 mg/L for posaconazole, 0.125 mg/L for caspofungin and 0.03 mg/L for anidulafungin. Agreement within two 2-fold dilutions between MHIC (determined by microcalorimetry) and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. This proof-of-concept study demonstrated the potential of isothermal microcalorimetry for growth evaluation of Aspergillus spp. and real-time antifungal susceptibility testing.     

Comparison of Sensititre YeastOne with the NCCLS M38-A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus

The in vitro activities of amphotericin B, itraconazole, and voriconazole against 279 clinical isolates of Aspergillus fumigatus were studied by comparing Sensititre YeastOne with the reference method NCCLS (CLSI) M38-A. The methods were considered to agree when the results of the MICs by Sensititre were within 2 dilutions of the MICs obtained by NCCLS M38-A. Agreement of readings at 24, 48, and 72 h was as follows: amphotericin B (0%, 31.5%, 82.1%), itraconazole (86.7%, 98.3%, 98.2%), and voriconazole (92.8%, 98.3%, 98.9%). Sensititre YeastOne is a suitable alternative for the determination of the MICs of itraconazole and voriconazole against A. fumigatus when results are read at 48 h of incubation.     

In vitro susceptibilities of zygomycetes to conventional and new antifungals

In vitro susceptibilities of 36 zygomycete isolates, belonging to six genera, to itraconazole, posaconazole, voriconazole, terbinafine, amphotericin B and 5-fluorocytosine were determined by using a broth microdilution adaptation of the National Committee for Clinical Laboratory Standards M-38P reference method. The influence of incubation time on MIC values, and the performance of a spectrophotometric method for MIC determination in comparison with the visual reference method, were also evaluated. Amphotericin B was active against most of the isolates. All the isolates were highly resistant to 5-fluorocytosine (MICs > 256 mg/L). Voriconazole was significantly less active than the other drugs with an overall MIC(90) (MIC at which 90% of the isolates were inhibited) of 32 mg/L. In contrast, posaconazole showed good activity (MIC(90) 1 mg/L). A wide range of MICs, from 0.03 to > or =32 mg/L, was obtained for itraconazole and terbinafine. Differences in susceptibility between and within genera were noted. Rhizopus spp. were significantly less susceptible to itraconazole, posaconazole, terbinafine and amphotericin B than Absidia spp., and less susceptible than Mucor spp. to amphotericin B. Terbinafine appeared to be more active against Rhizopus microsporus than against Rhizopus oryzae (geometric mean MIC of 0.15 and 64 mg/L, respectively). The activity of the drugs was dependent on the incubation period. A significant increase in MICs was noted between 24 and 48 h of incubation. On the other hand, the two methods used for MIC determination (visual and spectrophotometric readings) showed good agreement. These results suggest that the zygomycetes are a heterogeneous group for antifungal susceptibility. Some of the conventional and new antifungals are effective in vitro; their efficacies in vivo remain to be determined. The spectrophotometric method appears to be a valuable alternative to the visual method for MIC determination for zygomycetes.     

In vitro activity of the new triazole BMS-207147 against Aspergillus species in comparison with itraconazole and amphotericin B

The in vitro activity of BMS-207147 against 80 clinical isolates of Aspergillus was compared with that of itraconazole and amphotericin B, using a validated microtiter method. Geometric mean MICs (in microg/ml) were as follows: 1.71 for BMS-207147, 0.67 for itraconazole, and 0.63 for amphotericin B. The range of concentrations of each drug was 0.125 to >16 microg/ml. Aspergillus fumigatus was significantly more susceptible to BMS-207147 (P < 0. 05) than A. terreus and A. flavus. No BMS-207147-resistant A. fumigatus isolates were identified, though eight itraconazole-resistant (MIC, >8 microg/ml) isolates were. BMS-207147 is active against Aspergillus spp. at slightly high concentrations compared with itraconazole and amphotericin B.