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.     

Evaluation of the microdilution, Etest and disk diffusion methods for antifungal susceptibility testing of clinical strains of Trichosporon spp

Trichosporon spp are well recognized as pathogens capable of causing invasive disease. Despite the increasing frequency and severity of trichosporonosis, data on the antifungal susceptibility of Trichosporon spp. are limited and recommendations for in vitro testing of this fungus are not included in the guidelines of the National Committee for Clinical Laboratory Standards. The purpose of this study was to determine the in vitro susceptibility of clinical Trichosporon isolates to systemic antifungals. We evaluated the in vitro activity of amphotericin B, fluconazole, itraconazole and voriconazole against 27 clinical isolates of Trichosporon spp. (14 T. mucoides and 13 T. asahii) using NCCLS M27-A2 reference microdilution, Etest and disk diffusion methods. In the microdilution and Etest methods Trichosporon spp. demonstrated relatively high minimum inhibitory concentrations (MICs) for fluconazole (MIC90 4 and 6 microg/ml, respectively) and relatively low MICs for voriconazole (MIC90 0.125 and 0.125 microg/ml, respectively). MICs for amphotericin B determined on antibiotic medium 3 were lower (MIC90 0.06 microg/ml) than those on RPMI (MIC90 1 microg/ml). Observed agreements were 81-100% according to these drugs. Disk diffusion zone diameters correlate inversely with MICs from dilution tests except for amphotericin B. Validation of the clinical significance of these observations demands determination of MIC breakpoints for Trichosporon and in vitro- in vivo correlation studies.     

In vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and Cryptococcus gattii to five antifungal drugs

The in vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and C . gattii to five antifungal drugs (amphotericin B, flucytosine, fluconazole, itraconazole and ketoconazole) were determined using the Etest method. None of the Malaysian isolates was resistant to amphotericin B and ketoconazole. Isolates resistant to flucytosine, fluconazole and itraconazole were observed in this study. Minimum inhibition concentrations (MICs) of > or = 32 microg ml(-1) against flucytosine, > or = 64 microg ml(-1) against fluconazole and > or = 1 microg ml(-1) against itraconazole were noted in four (8.3%), two (4.2%) and one (2.1%) isolates respectively. There was no significant difference in the MICs for both Cryptococcus species (P > 0.05), indicating that C. gattii was as susceptible as var. grubii to all the antifungal drugs tested. No significant difference in the MICs for both Cryptococcus species collected from 1980 to 1990 and 2002 to 2004 were observed (P > 0.05).     

Antifungal drug susceptibility of Cryptococcus neoformans from clinical sources in Nairobi, Kenya

The serotypes and mating types of 80 clinical isolates of Cryptococcus neoformans from Kenya were studied and subjected to broth microdilution susceptibility testing to amphotericin B (AMP), flucytosin, fluconazole (FLC), itraconazole (ITC) and miconazole (MCZ). The isolates included C. neoformans var. grubii- 75 of 80 (serotype A; 93.7%), C. neoformans var. neoformans- three of 80 (3.8%) and C. neoformans var. gattii- two (serotype B; 2.5%). Mating experiment confirmed all the isolates to be alpha-mating type. Seventy-eight (97.5%) of the isolates had minimum inhibitory concentration (MIC) of < or =0.5 microg ml(-1) to AMP and at 1 microg ml(-1), 100% of the isolates were inhibited. Flucytosin resistance was observed in 21% with MIC in which 90% of the isolates were inhibited (MIC90) of 64 microg ml(-1). Only 23.8% of the strains were susceptible to FLC with 65% susceptible dose-dependent (SDD) and 11.2% resistant. Itraconazole susceptibility was 61.3% while the rest were either SDD or resistant. The MIC90 for ITC and MCZ were 0.5 and 2 microg ml(-1) respectively. The study reports the serotypes, mating types and highlights the existence of azoles resistance in C. neoformans in Nairobi which calls for antifungal drug resistance surveillance as prophylactic use of FLC increases because of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in sub-Saharan Africa.     

In vitro activities of new and conventional antimycotics against fluconazole-susceptible and non-susceptible Brazilian Candida spp. isolates

The substantial increase in the rate of azole resistant Candida spp. yeast infections has become a serious treatment problem requiring new and more active antifungal agents. In this study, the in vitro activities of ravuconazole and albaconazole were compared with those of amphotericin B, flucytosine, itraconazole and fluconazole against 162 Brazilian isolates of Candida spp. from which 48 isolates had previously shown lower susceptibility or resistance to fluconazole. Ravuconazole susceptibility ranged from 84.6% (Candida albicans) to 100% for other species and albaconazole MIC(90) was < or =1.0 microg ml(-1) for all the species emphasising the potent activity of these triazoles. To our knowledge this is the first study evaluating the susceptibility of C. dubliniensis to albaconazole.     

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.     

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 susceptibility of Cryptococcus neoformans serotypes to GM 237354 derivative of the sordarin class

In vitro susceptibility to the sordarin derivative GM 237354 and amphotericin B were tested in a total of 190 Cryptococcus neoformans clinical isolates from different geographical areas of Spain and South American countries. Minimal inhibitory concentrations (MICs) were obtained using the NCCLS reference microbroth dilution method and analysed according the serotypes of Cr. neoformans. The MICs for amphotericin B were lower than 1.0 microg ml(-1) (MIC90% 0.5 microg ml(-1) , MIC50% 0.125 microg ml(-1)) but five isolates showed MICs of 2.0 microg ml(-1) to GM 237354 (MIC90% 1.0 microg ml(-1), MIC50% 0.5 microg ml(-1)). Cryptococcus neoformans var. gattii serotype B, was significantly less susceptible than A and AD serotypes (P = 0.047 and P = 0.022, respectively).     

Fluconazole and itraconazole susceptibilities of Candida spp. isolated from oropharyngeal specimens and blood cultures of paediatric haematology/oncology patients

This study examined the in vitro susceptibilities to fluconazole and itraconazole of isolates of Candida spp. from surveillance oropharyngeal specimens and blood cultures from paediatric patients with malignancy. The species distribution of 100 isolates from oropharyngeal specimens was C. albicans 86%, C. glabrata 7%, C. lusitaniae 4%, C. parapsilosis 2% and C. tropicalis 1%. From a total of nine isolates from blood cultures the species distribution was C. albicans 33.3%, C. parapsilosis 33.3 % and C. guilliermondii 33.3%. Only three of the oropharyngeal isolates were resistant to fluconazole (MIC > or = 64 mg l(-1)) and only two were resistant to itraconazole (MIC > or = 1 mg l(-1)). None of the blood culture isolates was resistant to either agent. At this centre, C. albicans is the predominant species from oropharyngeal specimens, but non-albicans Candida species predominate in blood cultures. Although resistance to fluconazole and itraconazole is rare at present, continued surveillance is warranted to monitor trends in species distribution and antifungal susceptibility.     

Susceptibility profile and epidemiological cut‐off values of Cryptococcus neoformans species complex from Argentina

Epidemiological cut‐off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild‐type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC₅₀, MIC₉₀ and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml^?1 each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml^?1 respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested.     

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 antifungal susceptibility testing of filamentous fungi with Sensititre Yeast One

Sensititre is a colorimetric microdilution method for in vitro antifungal susceptibility testing based on the M27-A document (National Committee for Clinical Laboratory Standards) for yeasts. Difference between both methods is the presence of Alamar-blue and RPMI 1640 (glucose 2%) as culture medium. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine, 100 opportunistic filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp.) obtained from pathological samples was determined by the Sensititre method. Induction to conidium and sporangiospore formation at 35 degrees C was used to get inoculum and plates were covered by 1 ml of saline and suspensions were made by gently probing by a sterile loop. Optical densities of the conidial suspensions were adjusted to 80-82% transmittance for Aspergillus spp. and 68-70% for the rest of strains tested. Final inoculum concentration size was 0.4 x 10(4)-5 x 10(4) CFU ml(-1). Readings were made at 72 h of incubation at 35 degrees C; amphotericin B and itraconazole was active against Aspergillus fumigatus with CMI90 1 and 0.5 microg ml(-1), respectively, opposite to Scedosporium prolificans and Scedosporium apiospermum. As it was expected, a CMI90 of 256 microg ml(-1) for fluconazole and CMI90 for flucytosine amounting to 64 g ml(-1) were obtained. Sensititre Yeast One is a useful method and an alternative to reference methods to determine antifungal susceptibility of filamentous fungi for clinical laboratory routine. Correlation with microdilution results is studied. New triazole derivatives should be included as soon as their clinical use will be feasible.     

Susceptibility to antifungal agents of Candida species isolated from paediatric and adult patients with haematological diseases

Summary The susceptibility to six antifungals: amphotericin B (AMF), 5-fluorocytosine (5-F), miconazole (MIK), ketoconazole (KET), fluconazole (FLU) and itraconazole (ITR) was tested among 206 Candida spp. isolated from paediatric and adult patients with haematological malignancies. To determinate the susceptibility the commercial microdilution method Fungitest (Bio-Rad, France) was used. The strains were classified as susceptible, intermediate susceptible, or resistant on the base of the growth in following breakpoint concentrations of particular drugs: 2 and 8 microg ml(-1) for AMF, 2 and 32 microg ml(-1) for 5-F, 0.5 and 8 microg ml(-1) for MIK, 0.5 and 4 microg ml(-1) for KET and ITR, and 8 and 64 microg ml(-1) for FLU. The highest activity to overall species showed AMF (only one resistant strain) and 5-F (85% susceptible strains). Most of C. albicans isolates were susceptible to tested azoles. The percentages of C. albicans resistant to FLU, ITR, KET and MIK were 4, 11, 8, and 0.8%, respectively. The less susceptible to azoles were C. glabrata and C. krusei (14% and 44% isolates resistant to FLU). A non-albicans Candida isolated from adult patients receiving KET prophylaxis was more frequently resistant to FLU than isolates from patients without previous exposure to azoles (P < 0.05). We did not observe differences in the susceptibility of Candida strains isolated from children compared with those from adults.     

Determination of minumum inhibitory concentrations of Candida species isolated from vaginal swab specimens by using broth macrodilution and E-test

The purpose of the present study was to evaluate the utility of the E-test in determining the antifungal susceptibility of Candida species. A total of 50 Candida strains, including 34 Candida albicans and 16 non-albicans were isolated from vaginal swab specimens from women suffering from vaginitis. The minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole and ketoconazole were detected by using broth macrodilution and the E-test. When the results of the two tests were compared, the MIC values were considered acceptable if the difference between the two assays was no more than two-fold (+/-1dilution). The acceptable rates were: 84% for amphotericin B, 97% for fluconazole and 78% for ketoconazole. Finally, MICs of C. albicans against the tested antifungal agents were generally lower than for non-albicans strains. These results suggest that the E-test can be used for the determination of MIC values for Candida species isolates.     

In Vitro Susceptibility of Trichosporon beigelii to Antifungal Agents

Abstract

The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B, flucytosine, miconazole, fluconazole and itraconazole against 21 isolates of Trichosporon beigelii in RPMI-1640 medium were determined using National Committee for Clinical Laboratory Standards (NCCLS) methodology in microdilution method. Most isolates were sensitive to miconazole (MIC₉₀ 0.78 μ/ml), fluconazole (MIC₉₀ 6.25 μ/ml), and itraconazole (MIC₉₀ 0.19 μ/ml), with the former being the most active agent tested (MFC₉₀ 3.12 μ/ml). Although amphotericin B inhibited most strains (MIC range, 0.78-3.12 μ/ml), poor fungicidal activity was observed (MFC range, 1.56 -12.5 μ/ml) showing a pattern of relative resistance In Vitro. Flucytosine showed generally poor activity against most isolates tested. These In Vitro findings confirm the resistance of T.beigelii to amphotericin B and suggest that azoles may be an alternative to the former for the treatment of disseminated trichosporonosis. However, in vivo studies would better validate these In Vitro findings.     

Impact of endpoint definition on the outcome of antifungal susceptibility tests with Candida species: 24- versus 48-h incubation and 50 versus 80% reduction in growth

The growth inhibition patterns of 764 clinical yeast isolates, in response to amphotericin B, flucytosine, itraconazole and fluconazole, were studied in order to determine the frequency of trailing growth and any impact this, as well as 24- or 48-h incubation periods, may have on minimum inhibitory concentration (MIC) results. A broth microdilution method following National Committee for Clinical Laboratory Standard No. M27A recommendations was used. Trailing growth was observed mainly with azoles. Furthermore, over 98% of isolates exhibiting a trailing effect at 24 h with fluconazole and itraconazole were either Candida albicans or Candida tropicalis. When comparing 24- and 48-h IC50 values, discrepancies were observed with itraconazole and fluconazole, respectively, in 18 and 11% of C. albicans and 24 and 30% of C. tropicalis. When comparing IC50 and IC80 values at 24 h, discrepancies were again essentially seen with itraconazole and fluconazole, respectively, in 11 and 10% of C. albicans, and 17 and 27% of C. tropicalis. In susceptibility tests performed with a microdilution method and read spectrophotometrically, 48-h IC80 values result in an unlikely high number of resistant isolates, indicating that a 24-h incubation and a 50% reduction in optical density may correlate better with clinical outcome.     

Evaluation of semisolid agar screening tests for determining fluconazole and amphotericin B susceptibilities of Candida strains by using three different media

The susceptibilities of 164 Candida isolates against fluconazole and amphotericin B were determined by semisolid agar screening tests and the microdilution method according to NCCLS M27-A standards. The semisolid agar screening tests were performed with three different media containing 0.5% agar and 2, 8, and 40 microg/ml of fluconazole or 0.5 and 2.0 microg/ml of amphotericin B. These media were MOPS buffered RPMI 1640, brain-heart infusion and 1/3 diluted Sabouraud dextrose agar. The results of both methods were interpreted as susceptible, dose dependent susceptible and resistant for fluconazole and susceptible and resistant for amphotericin B. The agreement rates of semisolid agar screening tests using RPMI 1640, brain-heart infusion and Sabouraud dextrose media with the reference microdilution method were found to be 71.4%, 51.2%, and 57.3% for fluconazole and 79.3%, 53.7%, and 56.7% for amphotericin B, respectively. Overall, we conclude that semisolid agar screening tests using RPMI 1640 can be used for determining the susceptibilities of Candida isolates against fluconazole and amphotericin B in clinical microbiology laboratories.     

Effect of ketoconazole, itraconazole and fluconazole on the gastrointestinal colonization of mice by Candida albicans

Crl:CD1 (ICR) BR mice were colonized in the gastrointestinal (GI) tract with Candida albicans. This strain was susceptible to ketoconazole (MIC=0.25 microg/ml), itraconazole (minimum inhibitory concentration, MIC=0.25 microg/ml), and fluconazole (MIC=4 microg/ml). Subsequently the animals received monotherapy with ketoconazole by mouth (equivalent to human dose of 2.9 mg/kg/day), or itraconazole by mouth (equivalent to human dose of 2.9 mg/kg/day), or fluconazole either subcutaneously (equivalent to human dose of 2.2 mg/kg/day), or by mouth (equivalent to human dose of 2.2 mg/kg/day), for 10 days. Quantitative stool cultures at the end and one week after the end of treatment revealed that all three azoles caused a small and statistically non significant reduction of C. albicans concentration in the stools. The different route of administration of fluconazole did not produce different results. In conclusion, these azoles, used at the present doses and schedules, have minimal effect on murine GI colonization by this strain of C. albicans which is susceptible but with rather increased MICs.     

In vitro susceptibility of 545 isolates of Candida spp. to four antifungal agents

Summary. The in vitro susceptibility to amphotericin B, fluconazole, itraconazole and ketoconazole of 545 Candida strains from patients treated at the University Hospital of the Canaries was determined by means of a microdilution test. The distribution of the species was as follows: Candida albicans (342), Candida tropicalis (70), Candida glabrata (68), Candida parapsilosis (65). Of Candida albicans isolates, 8.5% and 7.6% showed resistance to itraconazole and fluconazole respectively. Of C. tropicalis isolates 34.3%, 27.1% and 2.9% were resistant to itraconazole, fluconazole and ketaconazole respectively. For C. glabrata , 10.3% and 4.4% of the isolates under study demonstrated resistance to fluconazole and itraconazole respectively. Only 4.6% and 1.5% of C. parapsilosis isolates demonstrated resistance to fluconazole and itraconazole respectively. C. tropicalis was the most resistant strain and C. parapsilosis the most sensitive. The greatest percentages of resistance in vitro were seen with the triazoles.
Zusammenfassung. Es wurde die Empfindlichkeit von 545 Candida -Stämmen für Amphotericin B, Fluconazol, Itraconazol und Ketoconazol in vitro in einem Mikrodilutionstest bestimmt. Bei den Stämmen handelte es sich um Isolate von Patienten, die in der Universitäts-Klinik der Kanarischen Inseln behandelt worden waren. Das Untersuchungsgut war wie folgt verteilt: 342 Candida albicans , 70 C. tropicalis , 68 C. glabrata , 65 C. parapsilosis. Bei C. albicans waren 8.5% gegen Itraconazol und 7.6% gegen Fluconazol resistent. Bei C. tropicalis wurden 34.3% resistent gegenüber Itraconazol befundet, 27.1% gegen Fluconazol und 2.9% gegen Ketoconazol. Bei C. glabrata waren 10.3% resistent gegen Fluconazol und 4.4% gegen Itraconazol. Candida parapsilosis wurde zu 4.6% gegen Fluconazol und zu 1.5% gegen Itraconazol als resistent befundet. Somit erwies sich C. tropicalis als die resistenteste und C. parapsilosis als die sensibelste Art.     

Skin infection caused by Scedosporium apiospermum

A woman with a skin infection because of Scedosporium apiospermum, in the interdigital spaces of her feet is presented. The minimum inhibition concentration values (MIC, microg ml(-1)) of this isolated mould for itraconazole, amphotericin B and terbinafine after 48 h were determined as 1, 8 and 16, respectively. The patient was treated successfully with oral terbinafine and topical clotrimazole.