In vitro activity (MIC and MFC) of voriconazole, amphotericin B, and itraconazole against 192 filamentous fungi: the GISIA-2 study

We evaluated the in vitro activity of voriconazole, amphotericin B, and itraconazole against 192 clinical mould isolates recovered in twenty Italian microbiology laboratories. The vast majority of isolates belonged to the genus Aspergillus (94.2%) with A. fumigatus (58.3%) being the most frequently isolated species. Antifungal susceptibility testing was performed using the broth microdilution method defined by the CLSI M38-A standard, and results were compared to those obtained with Sensititre panels. Aspergillus flavus ATCC 204304 was employed as reference strain and results were within all expected ranges. Voriconazole's activity against the 192 mould isolates was comparable to that of amphotericin B and itraconazole: voriconazole MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml), itraconazole MIC90 (CLSI 0.5 microg/ml, Sensititre 0.5 microg/ml), amphotericin B MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml). In conclusion, these in vitro data highlight voriconazole's broad spectrum activity against filamentous fungi and support its use as a first line agent for the treatment of fungal diseases.     

In vitro testing of Aspergillus fumigatus clinical isolates for susceptibility to voriconazole, amphotericin B and itraconazole: comparison of sensititre versus NCCLS M38-A using two different inocula

Voriconazole, amphotericin B and itraconazole were tested in vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol-NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 microg/ml for voriconazole, 0.06 to 1 microg/ml for amphotericin B, 0.016 to >16 microg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC90 values obtained with the two testing methods or with the two types of inocula. These findings confirm the good in vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation.     

In vitro studies on the activity of amphotericin B and lipid-based amphotericin B formulations against Aspergillus conidia and hyphae

The minimum inhibitory concentrations (MICs) of amphotericin B and lipid-based amphotericin B formulations against isolates of Aspergillus spp. were tested using a broth microdilution method. Twelve isolates of Aspergillus fumigatus, eight of Aspergillus flavus, six of Aspergillus niger and seven of Aspergillus terreus were examined. In addition, an assay for hyphae of Aspergillus spp. was performed since the invasive form is manifested by the appearance of hyphal structures. MICs of hyphae against lipid-based amphotericin B formulations were within three dilutions higher than those against conidia for almost all isolates of Aspergillus spp. (P < 0.01). In contrast, the differences in the in vitro efficacies of amphotericin B were the lowest for Aspergillus spp. This study demonstrates the importance of the type of inoculum used to test antifungal susceptibilities of Aspergillus spp. The significance of these results for in vivo outcome needs to be determined.     

Comparison of disk diffusion, E-test and broth microdilution test in determination of susceptibility of Aspergillus species to amphotericin B, itraconazole and voriconazole

In response to the recent increase in Aspergillus infections, new antifungal agents have become available accompanied by studies on antifungal susceptibility tests for epidemiological follow-up. The aim of this study was to compare the efficacy of Clinical Laboratory Standards Institute (CLSI) M 38-A broth microdilution test with the disk diffusion and E-test in determining the susceptibility of Aspergillus spp. to amphotericin B, itraconazole and voriconazole. The study was carried out on 18 A. fumigatus, 7 A. flavus, 5 A. niger and 2 A. versicolor strains isolated from clinical samples. The microdilution method was performed by following the instructions of CLSI M 38-A. The E-test and disk diffusion tests were performed according to the instructions of their manufacturers. The percent agreement between the E-test and CLSI M38-A broth microdilution test at 24 (48) h within +/- 2 dilutions was, respectively, 81% (69%) for amphotericin B, 75% (72%) for itraconazole and 85% (81%) for voriconazole. The disk diffusion test showed good correlation with the E-test but poor correlation with the broth microdilution test for the three antifungal agents we tested. In conclusion, E-test and disk diffusion test have their advantages such as ease of application and interpretation, but their correlation with the broth microdilution should be improved.     

Species assignment and antifungal susceptibilities of black aspergilli recovered from otomycosis cases in Iran

Black aspergilli are among the main causative agents of otomycosis worldwide. In this study, the species assignment of black aspergilli isolated from otomycosis cases in Iran was carried out using sequence analysis of part of the calmodulin gene. The results indicate that Aspergillus niger is not the only black Aspergillus species involved in otomycosis cases in Iran: Aspergillus awamori and Aspergillus tubingensis are also able to cause ear infections. Antifungal susceptibility tests were carried out against five antifungal drugs including amphotericin B, fluconazole, itraconazole, ketoconazole and terbinafine. All isolates were highly susceptible to terbinafine, while they exhibited moderate susceptibilities against amphotericin B, fluconazole and ketoconazole. Aspergillus niger and A. awamori were found to have higher minimal inhibitory concentrations for azoles than A. tubingensis, in accordance with previous findings.     

Case Reports. Chronic and acute Aspergillus meningitis

Cerebral aspergillosis usually occurs in severely immunocompromized hosts, is difficult to diagnose, and has a poor prognosis. After 14 months of chronic meningitis, ventriculitis, choroid plexitis, and lumbar arachnoiditis, which was complicated by acute hydrocephalus, Aspergillus, suspected to be from the candidus group, was isolated from the cerebrospinal fluid (CSF) of a previously healthy man. Thereafter Aspergillus antigen was found in stored plasma and CSF samples. He was treated with voriconazole and itraconazole. In a haemodialysis patient affected by an acute meningococcal meningitis, following a 3-day symptom-free interval, symptoms and signs of acute meningitis had reappeared and were unresponsive to a broad antimicrobial coverage. However, they resolved within 5 days after liposomal amphotericin B treatment had been started. From his CSF Aspergillus-DNA was identified and Aspergillus fumigatus isolated by culture. These two different clinical cases show that Aspergillus-DNA and antigen detection tests represent an advance in the diagnosis and liposomal amphotericin B, voriconazole, and itraconazole are an advance in the treatment of Aspergillus meningitis.     

Emergence of resistance to amphotericin B and triazoles in Candida glabrata vaginal isolates in a case of recurrent vaginitis

Emergence of resistance to triazoles and amphotericin B in Candida glabrata vaginal isolates is documented by Etest. During the 18-month follow-up of a case of vaginitis, 14 consecutive isolates of C. glabrata were examined. The isolates exhibited development of in vitro resistance beginning with itraconazole (>32 microg/ml), followed by fluconazole (>256 microg/ml), amphotericin B (>32 microg/ml), and voriconazole (>32 microg/ml). The DNA sequence analyses and finger printing of the isolates strongly suggest that our patient remained colonized with a single strain. The report underscores the propensity of C. glabrata to acquire resistance during antifungal therapy and the importance of susceptibility testing in the management of infections caused by this species.     

In-vitro activities of terbinafine,itraconazole and amphotericin B against Aspergillus and Cladosporium species

The in vitro fungicidal and fungistatic activities of terbinafine were compared with those of itraconazole and amphotericin B against Aspergillus (n=63) and Cladosporium (n=21) isolates. The broth macrodilution modification of NCCLS reference method for filamentous fungi (M38-P) was used to assess the minimum inhibitory concentrations (MICs). Our data show that the in vitro activities of terbinafine were comparable to those of itraconazole and amphotericin B against the Aspergillus spp and Cladosporium strains tested. Despite strain-dependent variabilities, in general, itraconazole's activity was similar to that of amphotericin B against strains of Aspergillus and Cladosporium. Our data suggest that terbinafine may be useful in the treatment of Aspergillus and Cladosporium infections.     

In vitro antifungal activity of amphotericin B and 11 comparators against Aspergillus terreus species complex

Aspergillus terreus infections are difficult to treat because of the intrinsic resistance to amphotericin B, and higher mortality compared to infections caused by other Aspergillus species. The aim of the present study was to determine the in vitro antifungal activity of amphotericin B and 11 comparators against clinical (n = 36) and environmental (n = 45) A. terreus isolates. In vitro antifungal susceptibility was performed using the CLSI M38‐A2 procedure. Amphotericin B exhibited the highest MICs (MIC range, 0.125‐4 μg/mL; MIC₉₀, 2 μg/mL), followed by terbinafine (MIC range, 0.002‐1 μg/mL; MIC₉₀, 1 μg/mL). Only one isolate (1/81) showed amphotericin B MIC above the epidemiologic cut‐off value (ECV; 4 μg/mL). None of the isolates had a MIC of ≥ ECV for voriconazole, itraconazole and posaconazole. The reasons for the difference in amphotericin B susceptibility patterns between studies remain unknown. The genetic and species diversity, clinical, environmental and ecological factors in Terrei section on various amphotericin B susceptibility profiles in different countries should be considered more as the main reasons associated with these differences.     

Molecular typing and antifungal susceptibility of clinical and environmental Cryptococcus neoformans species complex isolates in Goiania, Brazil

A total of 124 Cryptococcus isolates, including 84 clinical strains obtained from cerebrospinal fluid from AIDS patients and 40 environmental isolates from pigeon excreta and from Eucalyptus trees, were studied. The varieties, serotypes, phospholipase activity and molecular profile of these isolates were determined. Cryptococcus neoformans var. grubii serotype A was identified in 120 isolates and Cryptococcus gattii serotype B in four isolates. The clinical isolates showed higher phospholipase activity than environmental isolates. Similar patterns of in vitro susceptibility to amphotericin B, fluconazole, itraconazole and voriconazole and no resistance were found for all isolates. Molecular type VNI ( C. neoformans var. grubii ) was recovered in 80 clinical and 40 environmental isolates while the type VGIII ( C. gattii ) was found in four clinical isolates. This study demonstrated for the first time the molecular types of clinical and environmental Cryptococcus isolates in the midwest Brazil region.     

Genetic re-identification and antifungal susceptibility testing of Aspergillus section Nigri strains of the BCCM/IHEM collection

Black Aspergilli are widely distributed in the environment and are frequently reported as causative agents of different types of mycoses. Many taxonomical revisions have been made, and presently 19 different species are accepted. In this study we (re-) identified 123 strains of the Aspergillus niger group of the BCCM/IHEM collection to check for the presence of species other than A. niger in both environmental and clinical samples. The susceptibility for antifungal drugs was compared between A. niger and Aspergillus tubingensis. Strains were identified based on morphological and molecular data and neighbour joining analysis. We revealed the presence of eight different species of this group in our collection. Our results suggest that Aspergillus foetidus, previously shown to be a species closely related to A. niger should not be considered as a separate species, but rather as a variety of A. niger. Furthermore, we found A. tubingensis at the same prevalence than A. niger in clinical samples. Interestingly, A. niger was shown to have a twofold higher sensitivity to treatment with voriconazole and itraconazole than A. tubingensis. These findings underline once more the importance of correct identification up to the species level in clinical isolates.     

In vitro susceptibility of 188 clinical and environmental isolates of Aspergillus flavus for the new triazole isavuconazole and seven other antifungal drugs

Recently isavuconazole, an experimental triazole agent, was found to be active against Aspergillus species. As Aspergillus flavus is the second-most common Aspergillus species isolated from human infection and the fungus has not been widely tested against the drug, we studied a large collection of clinical (n = 178) and environmental (n = 10) strains of A. flavus against isavuconazole and compared the results with seven other Aspergillus-active antifungal agents (some of them triazoles, others echinocandins or polyene antifungals: voriconazole, posaconazole, itraconazole, caspofungin, anidulafungin, micafungin and amphotericin B) using Clinical and Laboratory Standards Institute methods. Strains with high minimal inhibitory concentrations (MICs) were tested by E-test as well. The strains were collected from two different geographical locations (India and the Netherlands). Three isolates (1.6%) had high MIC (2 mg l(-1) by microbroth dilution and 8 mg l(-1) by E-test) for amphotericin B. Isavuconazole showed good activity against A. flavus strains with MIC(50) and MIC(90) values of 1 mg l(-1). As compared with voriconazole (the drug recommended for primary therapy of aspergillosis), isavuconazole had better activity (99.5% of strains had MIC of ≤ 1 mg l(-1) for isavuconazole, compared to 74% of strains with same MIC for voriconazole). All strains were, following recently proposed clinical breakpoints, susceptible for the triazoles tested except three strains, which had MICs of 4 mg l(-1) for voriconazole. Testing these strains with high MIC by E-test, gave results of 0.5-2 mg l(-1). Posaconazole had the lowest MIC(50) and MIC(90) of 0.125 mg l(-1) and 0.25 mg l(-1), respectively. Among echinocandins, 97% of strains had a minimum effective concentration (MEC) of ≤ 0.5 mg l(-1) for caspofungin, and all strains had a MEC of ≤ 0.016 mg l(-1) and ≤ 0.125 mg l(-1) for anidulafungin and micafungin, respectively.     

In vitro activities of voriconazole (UK-109, 496), fluconazole, itraconazole and amphotericin B against 132 non-albicans bloodstream yeast isolates (CANARI study)

The aim was to evaluate the in vitro activity of voriconazole compared with those of amphotericin B, itraconazole and fluconazole against 132 bloodstream isolates of Candida non-albicans and Saccharomyces cerevisiae species. The minimal inhibitory concentrations (MICs) were determined by an adapted National Committee for Clinical Laboratory Standards (NCCLS) M27-A method using RPMI 1640 as test medium supplemented with 2% glucose. MIC end-points were determined with a spectrophotometer after incubation for 48 h at 35 degrees C. Optical density data were used for the calculation of the MIC end-points. For amphotericin B, the end-point was defined as the minimal antifungal concentration that exerts 90% inhibition compared with the control well growth. For the azoles, the end-points were determined at 50% inhibition of growth. Amphotericin B is highly active with 97% of isolates inhibited by < or =1 microg ml(-1). Decreased susceptibility or resistance to fluconazole was the rule among C. krusei, which is intrinsically resistant to fluconazole. For C. glabrata isolates, resistance to fluconazole and itraconazole was measured in 13% and 17% of the isolates respectively. Voriconazole was quite active in vitro against all the isolates with a MIC90% of < or =1 microg ml(-1) and we conclude that it may be useful in the treatment of non-albicans bloodstream infections.     

Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B‐resistant isolates

Aspergillus spp. are the most common invasive mould infection and are responsible for high mortality. Aspergillus fumigatus is currently of interest because resistance to azole antifungals has emerged. The Campinas University Hospital (HC ‐UNICAMP ) receives high‐risk patients susceptible to opportunistic infections but there have been no reports of resistant A. fumigatus . This study aimed to assess the susceptibility profile of Aspergillus isolates, specifically looking for azole resistance. ITS and β‐tubulin DNA sequencing was performed on 228 sequential clinical isolates. Broth microdilution susceptibility testing was performed for all isolates. A. fumigatus represented 74% of the isolates followed by Aspergillus flavus (12%). Nine A. fumigatus isolates from 9 different patients showed high MIC values to at least 1 azole, but cyp51A polymorphisms were detected in only 6 isolates and none correlated with known resistance mutations. The most troubling observation was that the minimum inhibitory concentration for amphotericin B was elevated (≥2 mg L^?1) in 87% of patients with A. flavus isolates and 43% with Aspergillus fumigatus isolates. Given that amphotericin B is used to treat azole‐resistant infections, these data highlight the need for continuous surveillance in Aspergillus for all antifungal resistance to implement correct treatment strategies for the management of these pathogens.     

In vitro interactions between amphotericin B and other antifungal agents and rifampin against Fusarium spp.

Fusarium species are common hyaline soil saprophytes and plant pathogens that are opportunistic fungal pathogens of immunocompromised patients. The treatment for fusariosis remains uncertain with an unfavourable prognosis; new possibilities for treatment, such as various synergistic drug interactions, must be uncovered. In this study, we evaluated the in vitro interactions of amphotericin B with caspofungin, ketoconazole, 5‐flucytosine, itraconazole, miconazole, rifampin, fluconazole, terbinafine and voriconazole against isolates of Fusarium spp. using the chequerboard method with interactions evaluated by fractional inhibitory concentration indices. The highest percentages of synergistic interactions were observed for the combinations of amphotericin B and caspofungin (68.7%), amphotericin B and rifampin (68.7%), amphotericin B plus 5‐flucytosine (59.3%) and amphotericin B with voriconazole (37.5%). The pattern of susceptibility to antifungal agents among Fusarium species and their consequence on the effects of drug combinations are also discussed.     

In vitro susceptibility of Cryptococcus neoformans clinical isolates from Egypt to seven antifungal drugs

The in vitro susceptibility of 29 clinical isolates of Cryptococcus neoformans to fluconazole, miconazole, itraconazole, ketoconazole, flucytosine, nystatin and amphotericin B was tested by broth and colorimetric microdilution methods. Most of the isolates showed uniform patterns of susceptibility to the used antifungal agents. Only three isolates exhibited resistance [fourfold or greater rise in the minimum inhibitory concentrations (MICs)] to the tested antifungal drugs. The MIC50 and MIC90 were 0.5-8 mg l(-1) for 5-flucytosine, 0.2-8.25 mg l(-1) for nystatin, 0.5-16 mg l(-1) for fluconazole and 0.2-12.5 mg l(-1) for miconazole. However, MIC50 and MIC90 were in narrow range for the clinical yeast isolates in both methods used and showed 0.5-1 mg l(-1) for amphotericin B and 0.016-0.25 mg l(-1) for both ketoconazole and itraconazole. The combination of fluconazole plus flucytosine showed greater synergistic and fungicidal activity compared with that of fluconazole plus amphotericin B or the use of individual drugs.     

In Vitro Testing of Aspergillus fumigatus Clinical Isolates for Susceptibility to Voriconazole,Amphotericin B and Itraconazole: Comparison of Sensititre versus NCCLS M38-A Using Two Different Inocula

Abstract

Voriconazole, amphotericin B and itraconazole were tested In Vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol- NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 μg/ml for voriconazole, 0.06 to 1 μg/ml for amphotericin B, 0.016 to >16 μg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC₉₀ values obtained with the two testing methods or with the two types of inocula. These findings confirm the good In Vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation.     

In vitro activity of terbinafine and itraconazole against Aspergillus species isolated from otomycosis

The minimum inhibitory concentrations (MIC, microg ml-1) of itraconazole and terbinafine against overall 34 Aspergillus isolates from the external ear canals with otomycosis have been determined with M38-P microdilution method suggested by National Committee for Clinical Laboratory Standards (NCCLS). MIC intervals in 48 h determined by taking MIC-2 value of itraconazole (the lowest drug concentration causing 50% inhibition of visible fungal growth) and MIC-0 value of terbinafine (the lowest drug concentration causing 100% inhibition of visible fungal growth) as a basis have been found as follows: 0.125-1 and 0.06-0.5 microg ml-1 for A. niger (22 strains), 0.06-0.25 and 0.06-0.125 microg ml-1 for A. flavus (10 strains), 0.125 and 0.125-0.5 microg ml-1 for A. terreus (two strains). It has been observed that both of the antifungal agents showed an in vitro activity against all Aspergillus species tested.     

Aspergillus terreus: an emerging amphotericin B-resistant opportunistic mold in patients with hematologic malignancies

BACKGROUND: Invasive aspergillosis (IA) has emerged as a common cause of morbidity and mortality among immunocompromised patients. At The University of Texas M. D. Anderson Cancer Center (Houston, TX), Aspergillus terreus is second to A. fumigatus as the most common cause of IA. In the current study, the authors compared the risk factors and outcomes associated with IA caused by A. terreus and IA caused by A. fumigatus. METHODS: The authors retrospectively reviewed the medical records of 300 patients who received care at our institution between 1995 and 2001 and who had cultures that were positive for Aspergillus infection, including 90 patients whose cultures were positive for A. fumigatus and 70 patients whose cultures were positive for A. terreus. RESULTS: Thirty-two patients with IA caused by A. terreus and 33 patients with IA caused by A. fumigatus were evaluated. The two groups were comparable in terms of age, gender, and underlying disease. Leukemia was the most common underlying malignancy (84%). More than 40% of patients in each group had undergone bone marrow transplantation. There was a trend toward a higher frequency of neutropenia among patients with IA caused by A. terreus (P = 0.12). IA caused by A. terreus was considered to be nosocomial in origin significantly more frequently compared with IA caused by A. fumigatus (P = 0.03). In vitro, A. terreus was found to be more resistant to amphotericin B (minimal inhibitory concentration [MIC90], 4.0 microg/mL) than to antifungal therapy (MIC90, 1.0 Hg/mL) in the isolates that were tested (< 50% of all isolates). The overall rate of response to antifungal therapy was 39% for patients with A. fumigatus infection, compared with 28% for patients with A. terreus infection (P = 0.43). CONCLUSIONS: Despite the decreased in vitro susceptibility of A. terreus (relative to A. fumigatus) to amphotericin B, the two groups within the current patient population had comparably poor responses to amphotericin B preparation and somewhat improved responses to posaconazole.     

Effect of medium composition on static and cidal activity of amphotericin B,itraconazole, voriconazole,posaconazole and terbinafine against Aspergillus fumigatus: a multicenter study

The effect of the medium composition on the fungistatic (MIC) and fungicidal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilution, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 microl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.