Comparative evaluation of Sensititre YeastOne vs. the NCCLS M27A protocol and E-test for antifungal susceptibility testing of yeasts

A recently developed microdilution method (Sensititre) YeastOne) may represent a valid alternative to the National Committee for Clinical Laboratory Standards (NCCLS) method for routine testing. The Medical Mycology Committee of the Associazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate its reproducibility and reliability compared with the NCCLS M27A protocol and the E-test. Nineteen strains each of Candida albicans and Ca. parapsilosis, isolated from systemic infections, were tested against amphotericin B, flucytosine, ketoconazole, itraconazole, and fluconazole. All the participating laboratories tested the YeastOne panels, while the E-test and the NCCLS method were performed by two laboratories each. Interlaboratory reproducibility showed a good correlation (from 95% for amphotericin B to 92.5% for flucytosine). The agreement between NCCLS and YeastOne ranged from 95 (ketoconazole and itraconazole) to 100% (amphotericin B and flucytosine), whereas the agreement between E-test and YeastOne ranged from 72.5 (fluconazole) to 100% (amphotericin B and flucytosine). The Sensititre YeastOne panels appear to be an excellent alternative to both the E-test and the NCCLS protocol for antifungal susceptibility testing.     

In vitro activity of nitroxoline against clinical isolates of Candida species

The in vitro antifungal activity of the quinoline nitroxoline has been compared with those of amphotericin B, flucytosine, and two azoles, miconazole and ketoconazole, against clinical isolates of Candida spp. A total of 186 isolates of 10 species of Candida and two culture collection strains were tested by an agar-dilution technique. Nitroxoline was highly active against Candida spp. MICs for nitroxoline ranged between 0.25-2 micrograms ml-1 for 186 representative strains. With MIC90 as the measure of antifungal activity, nitroxoline appeared to be superior to the imidazoles studied. Data for individual species of Candida revealed that the activities of nitroxoline and amphotericin B were generally just as effective against C. albicans, whereas flucytosine was the most active agent against Candida spp.     

Antifungal susceptibility testing of Candida albicans by flow cytometry

Antifungal susceptibilities of 28 Candida albicans isolates and two quality control strains to amphotericin B and fluconazole were determined by flow cytometry and microdilution method. Minimum inhibitory concentrations (MICs) obtained by flow cytometry were compared with the results obtained by The National Committee for Clinical Laboratory Standards Subcommittee (NCCLS) broth microdilution method. The agreement of results (within two dilution) obtained was found as 96 and 93% for amphotericin B and fluconazole, respectively. At least 24 h incubation was required for reading the microdilution assays. Four hours of incubation was required for fluconazole, whereas 2-h incubation was sufficient for amphotericin B to provide MIC by flow cytometry. Results of this study show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of Candida albicans isolates.     

Antifungal susceptibility and genotypes of Candida albicans strains from patients with vulvovaginal candidiasis

Studies of the genetic diversity of Candida albicans strains and the correlation between the antifungal susceptibility and gene diversity of C. albicans were carried out and the results were found to be inconsistent. To investigate antifungal susceptibility and genotypes of C. albicans strains from patients with vulvovaginal candidiasis (VVC), the genotypes of C. albicans in patients with VVC were studied using a recently developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of CAI microsatellite method and antifungal susceptibility was tested using E-test methods. Twenty-six genotypes were identified from 89 strains of C. albicans isolated from patients with VVC. Candida albicans isolates were susceptible to amphotericin B, flucytosine, ketoconazole and fluconazole. The dominant genotypes (A, B, C, D) account for 69.7% (62/89) of C. albicans . The resistant rate of C. albicans genotype B to itraconazole (ITR) and that of C. albicans non-genotype B strains were 66.7% (14/21) and 4.4% (3/68) respectively at P  < 0.05. We concluded that C. albicans genotype B from patients with VVC was more resistant to ITR.     

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.     

Phenotypic variation and antifungal susceptibility patterns of Candida albicans strains isolated from neutropenic patients

The aim of this study was to investigate the relationship between phenotypes of Candida albicans strains isolated from clinical specimens and the susceptibility of the strains to three antifungal agents, fluconazole, amphotericin B and flucytosine. Oropharyngeal, gastrointestinal and urogenital tract specimens were collected from 122 neutropenic patients who had received no previous prophylactic treatment. Each of 122 C. albicans strains recovered was found to express one of the six phenotypes: smooth, fuzzy, irregular, star, ring and stipple. The mean minimum inhibitory concentrations (MICs) of fluconazole was consistently higher for C. albicans strains expressing the stipple phenotype. The mean MICs for the six phenotypes of C. albicans strains ranged between 1.22 and 7.94 micrograms ml-1 for fluconazole, 0.99 and 2.55 micrograms ml-1 for amphotericin B and 1.23 and 1.83 micrograms ml-1 for flucytosine. The antifungal susceptibility of the stipple phenotype requires attention, especially in patients who are clinically unresponsive to fluconazole chemotherapy or in cases of life-threatening C. albicans infections of immunocompromised hosts. Long-term use of fluconazole may explain the outcome of the resistant stipple phenotype.     

In vitro activity of rilopirox against fluconazole-susceptible and fluconazole-resistant Candida isolates from patients with HIV infection

The in vitro antifungal activity of the new hydroxypyridone antimycotic rilopirox has been evaluated against 38 fluconazole-susceptible and -resistant clinical isolates of Candida albicans together with other Candida species isolated from patients with human immunodeficiency virus (HIV) infection and oropharyngeal candidosis. Minimum inhibitory concentrations (MICs) of both rilopirox and fluconazole were measured by a microdilution method using high-resolution medium supplemented with asparagine and glucose at pH 7.0. In comparison, an agar dilution technique was carried out for susceptibility testing of the antifungal agents. Rilopirox was found to be able to inhibit growth of all clinical yeast isolates. The rilopirox MICs at which 50% and 90% of strains were inhibited (MIC50 and MIC90 respectively), as determined by the microdilution method, were 4 and 8 micrograms ml-1 respectively. The highest MIC values for rilopirox using microdilution and the agar dilution method were 32 or 25 micrograms ml-1 respectively. On the other hand, for fluconazole, the MIC50 and MIC90 achieved were 0.5 and 128 micrograms ml-1, respectively, which means that the MIC90 value of fluconazole was 16-fold higher than that of rilopirox. Using the agar dilution technique, the MIC values of rilopirox were in the range 0.006-25 micrograms ml-1 with a median of 3.12 micrograms ml-1. For fluconazole, the MIC90 value was four-fold higher than that for rilopirox, indicating a considerable proportion of yeast strains with high MICs of 100 micrograms ml-1, suggesting in vitro resistance to this azole antifungal. All strains with diminished fluconazole susceptibility were susceptible to rilopirox. Even Candida krusei and Candida glabrata exhibited good in vitro susceptibility to rilopirox. Therefore, this new antifungal agent may be used as an alternative not only in the treatment of vaginal candidosis, but also in oropharyngeal Candida infections, e.g. in AIDS patients.     

Fluconazole, itraconazole and ketoconazole in-vitro activity. A comparative study.

We compared in-vitro activity of fluconazole, itraconazole and ketoconazole by evaluating their Minimal Inhibitory Concentrations (MICs) for 100 fungal strains isolated from different biological specimens of ARC/AIDS patients. A semisolid agar medium was used: this method is suitable for testing molds and yeasts, and can be applied to all azole antifungal drugs. Fluconazole had higher MICs than two other tested drugs, especially for Candida krusei strains; however it never had a MIC higher than 40 mg/l. Itraconazole and ketoconazole had MICs higher than 40mg/l for one Cryptococcus neoformans strain. There were no significant differences for itraconazole and ketoconazole among the tested strains.     

In Vitro Antifungal Activity of Saperconazole (R 66905) Against Candida and Torulopsis Die antimyzetische Aktivität von Saperconazol (R 66905) in vitro gegen Candida und Torulopsis

The susceptibility of several strains of Candida and Torulopsis to saperconazole, a new triazole antifungal compound, was compared to that of ketoconazole. The MICs of the two antifungal agents were determined against 70 strains of Candida albicans, 10 strains of C. guilliermondii, 10 strains of C. krusei, 10 strains of C. parapsilosis, 10 strains of C. pseudotropicalis, 10 strains of C. tropicalis and 15 strains of Torulopsis glabrata. The fungistatic activity was evaluated by the agar dilution method using BHI and casitone media after incubation for 48 hours at 28-30 degrees C. The in vitro activity of saperconazole was similar to that of ketoconazole for most of the Candida spp. tested except for C. krusei in particular. An MIC of less than or equal to 3.12 micrograms/ml for saperconazole was found with 92% of the C. albicans strains tested. In contrast, T. glabrata was more susceptible to ketoconazole.     

In vitro susceptibility of yeasts for fluconazole and itraconazole. Evaluation of a microdilution test

In vitro susceptibilities were determined for a total of 159 clinical isolates and 12 reference strains of yeasts belonging to different Candida species including 94 Candida albicans strains, and further genera such as Cryptococcus, Trichosporon, Geotrichum and Saccharomyces. Minimum inhibitory concentration (MIC) values for fluconazole and itraconazole were assessed using a microdilution technique with the semisynthetic high resolution (HR) medium supplemented with glucose and asparagine but without sodium hydrogen carbonate (pH 7.0), according to a proposal of the working group 'Clinical Mycology' of the German Speaking Mycological Society. Fluconazole MIC values for C. albicans were between 0.125 and > or = 128 micrograms ml-1. Thus, the median of 1 microgram ml-1 showed that the overall fluconazole susceptibility was good. As expected, Candida krusei (seven strains) exhibited diminished in vitro susceptibility with MIC values for fluconazole of 8 to 128 micrograms ml-1 with a median of 64 micrograms ml-1. Some Candida kefyr strains seemed to be less susceptible against fluconazole which was indicated by a MIC90 of 64 micrograms ml-1. Surprisingly, no Candida glabrata isolate exhibited a MIC value greater than 16 micrograms ml-1. Other Candida species, Trichosporon cutaneum, Geotrichum candidum and Saccharomyces cerevisiae showed low MICs to fluconazole. In vitro susceptibility testing of itraconazole revealed that all Candida species except C. albicans, but also Trichosporon cutaneum, Geotrichum candidum, and Saccharomyces cerevisiae exhibited acceptable low MIC values against itraconazole (0.03-2 micrograms ml-1). Their MIC90 values for itraconazole were in the close range between 0.125 and 2 micrograms ml-1. MIC values between 0.125 and 2 micrograms ml-1 were obtained, even for C. krusei strains. On the other hand, the range of C. albicans MICs was between 0.0125 and > or = 16 micrograms ml-1 with MIC50 and MIC90 values of 0.125 and > or = 16 micrograms ml-1, respectively, indicating that a considerable number of yeast strains have high MICs. The comparative evaluation of different experimental conditions revealed that there exists a marked influence both of inoculum size and incubation time on the results of susceptibility testing. Therefore, for routine usage 10(2) CFU ml-1 and 18-24 h incubation time for this microdilution method with HR medium are recommended.     

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.     

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 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).     

Fluconazole,Itraconazole and Ketoconazole In- Vitro Activity. A Comparative Study

Summary

We compared in-vitro activity of fluconazole, itraconazole and ketoconazole by evaluating their Minimal Inhibitory Concentrations (MICs) for 100 fungal strains isolated from different biological specimens of ARC/AIDS patients. A semisolid agar medium was used: this method is suitable for testing molds and yeasts, and can be applied to all azole antifungal drugs. Fluconazole had higher MICs than two other tested drugs, especially for Candida krusei strains; however it never had a MIC higher than 40mg/l.

Itraconazole and ketoconazole had MICs higher than 40mg/l for one Cryptococcus neoformans strain. There were no significant differences for itraconazole and ketoconazole among the tested strains.     

Identification of Candida species isolated from patients in intensive care unit and in vitro susceptibility to fluconazole for a 3-year period

Species level identification of Candida and antifungal susceptibility testing is not generally performed in routine laboratory practice. There is limited information about the distribution of Candida species and antifungal susceptibility in Turkey. In this study, we aimed at identifying Candida isolates to species level from various samples obtained from patients treated in an intensive care unit between 2002 and 2005 and to evaluate fluconazole susceptibilities of the isolates. A total of 320 Candida isolates obtained from 270 patients were identified by conventional methods and using API (Candida and/or 20C AUX) system. Antifungal susceptibility testing was performed by broth microdilution method. Candida albicans was isolated with the highest frequency (65.6%) followed by C. parapsilosis (11.3%), C. glabrata (8.8%) and C. tropicalis (7.8%). Of all the isolates, 92.9% revealed susceptibility to fluconazole. Susceptibility to fluconazole was highest for C. albicans followed by C. parapsilosis and C. glabrata. The MIC(90) values for C. albicans, C. parapsilosis, C. glabrata and C. tropicalis were 1, 2, 8 and 4 mug ml(-1) respectively. Fluconazole remains effective against both C. albicans and the majority of non-albicans Candida species. In this study, we determine the distribution of Candida species and evaluate the susceptibilities of the isolates, particularly for the azoles.     

In vitro antifungal susceptibility of clinical isolates of Candida spp. from hospitalized patients

A total of 122 Candida spp. strains, isolated from a group of 100 patients hospitalized in the Santa Casa de Misericordia of Belo Horizonte were assayed for in vitro susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine using a microbroth technique proposed by the National Committee for Clinical Laboratory Standards. In this study large variations were observed among minimum inhibitory concentration values depending on the species tested. The statistical analysis (Kruskal-Wallis test) showed that itraconazole and flucytosine were the more efficient antifungal drugs for most of species, and amphotericin B and fluconazole were the least efficient.     

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.     

Fluconazole vs. amphotericin B for the management of candidaemia in adults: a meta-analysis

The incidence of bloodstream infections caused by Candida species is rising. Few published studies have compared the efficacy of fluconazole with that of amphotericin B. We performed a meta-analysis of the prospective studies that compared fluconazole and amphotericin B for the treatment of candidaemia in adults. Data on total mortality, candidaemia-attributable mortality, efficacy, microbiological failure, and toxicity were extracted from eligible studies. All studies appeared homogeneous with respect to the outcome measures. Most patients were at relatively low risk for death as evidenced by the low average physiologic score and the lack of intense immunosuppression. The odds ratios (OR) of treatment with amphotericin B versus fluconazole and 95% confidence intervals (CI) were as follows: total mortality (OR, 1.06; CI, 0.89-1.25), candidaemia-attributable mortality (OR, 1.0; CI, 0.70-1.45), clinical response (OR, 1.14; CI, 0.93-1.39) and microbiological failure according to all Candida species (OR, 0.99; CI, 0.78-1.26). A trend favouring amphotericin B was seen in mycological eradication of non-albicans Candida species (OR, 0.70; CI, 0.47-1.06). Finally, amphotericin B was more toxic than fluconazole (OR, 2.94; CI, 2.14-4.4). In conclusion, fluconazole is as efficacious and less toxic than amphotericin B in stable, not severely immunosuppressed candidaemic patients at low risk for death. However, fluconazole may be less effective than amphotericin B in candidaemias caused by some non-albicans Candida species.     

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.     

Azole resistance in neonatal intensive care units in Argentina

The aim of this study was to determine the antifungal susceptibility profile and to detect resistant strains of yeast species isolated from neonates in Intensive Care Units. 92 strains isolated from 25 bloodstream cultures, 20 venous catheters, 23 suprapubic aspirations and 24 rectal swabs were studied. A Candida glabrata strain resistant to fluconazole was detected. Candida krusei appeared with its inherent resistance to fluconazole and showed cross-resistance to itraconazole. Two Candida albicans strains were resistant to azoles, one to itraconazole and the other to fluconazole with a high minimum inhibitory concentration (MIC) for itraconazole. All Candida tropicalis strains were susceptible to fluconazole but two of them showed resistance to itraconazole. The detection of resistant strains in neonates whom had not received previous antifungal therapy is noteworthy. The variations in the epidemiology of fungal infections observed and the antifungal resistance detected emphasize the importance of performing a regular surveillance to observe and to assess them.