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.     

Effect of culture media on in vitro susceptibility testing of fluconazole against some yeasts

Thirty clinical isolates, comprising six strains of Candida albicans, and four strains each of C. lusitaniae, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Torulopsis glabrata and Trichosporon beigelii were tested against fluconazole, using Sabouraud's dextrose (SD) broth and a high resolution (HR) medium (Pfizer Central Research, Inc.). The procedure was a standard tube (1 ml/tube) dilution, and C. albicans Y01 09 was included as a reference strain to monitor quality and reproducibility. Results indicated that the minimum inhibitory concentrations (MICs) for all isolates of C. albicans, C. lusitaniae, C. tropicalis, and Tr. beigelii were 100 micrograms ml-1 or greater in the SD medium. In the HR medium, however, the MICs for two isolates of C. albicans were 1.56 micrograms ml-1, in other four gave higher values (greater than 100 micrograms ml-1), and the results for C. lusitaniae and Tr. beigelii were in the range 1.56-3.12 micrograms ml-1. The MICs for C. tropicalis were unaffected (100 micrograms ml-1) by the medium used. All Cr. neoformans isolates yielded a uniform value (1.56 micrograms ml-1) in HR medium as compared to somewhat more variable results (MICs 0.39-1.56 micrograms ml-1) in SD broth. The MICs for T. glabrata in the SD and HR media were 3.12-12.5 and 6.25 micrograms ml-1, respectively. The data indicated that the HR medium is preferable for the in vitro susceptibility testing of C. albicans, C. lusitaniae and Tr. beigelii to fluconazole. The MICs for other yeasts were not affected by the culture medium. The reference C. albicans isolate yielded an MIC of 1.56 micrograms ml-1 throughout.     

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.     

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 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.     

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 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 comparative evaluations of the postantifungal effect: synergistic interaction between flucytosine and fluconazole against Candida albicans

In vitro comparative evaluations were performed to study the efficacy of combinations of flucytosine and fluconazole in producing a postantifungal effect (PAFE) on Candida albicans. Initial studies were done to determine MIC, FIC (fractional inhibitory concentration) and optimal PAFE parameters. A turbidometric method was used to measure yeast cell growth following exposure to different concentrations of the two drugs for periods of 0.5, 1 or 2 h at temperatures of 30 degrees C and 37 degrees C. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. Ten strains of C. albicans were then assayed (30 degrees C; 2 h exposure time) and a synergistic PAFE was evidenced with the two drugs at concentrations well below their individual MICs. PAFEs ranging from 3.8 to 10.5 h, which persisted for 1.2-2.5 h longer than those achieved with either agent separately, were evidenced when flucytosine and fluconazole were combined (flucytosine: fluconazole ratios of 1:16-1:32) at concentrations ranging from 0.024 to 0.098 micrograms ml-1 and 0.78 to 1.56 micrograms ml-1 respectively. The concentrations of each agent required to produce an optimal PAFE varied according to the C. albicans strain being assayed.     

Comparison of Broth Dilution and Semisolid Agar Dilution for In Vitro Susceptibility Testing of Cryptococcus neoformans

Summary

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution.

By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively.

Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.     

Oropharyngeal carriage of Candida species in HIV-infected patients in India

The present investigation represents the first study of oropharyngeal carriage of Candida and other yeasts in HIV-infected patients in India. One hundred and fifty HIV-positive patients were investigated by culturing their swish samples on plates of CHROMagar Candida. Ninety-eight patients (65.3%) were positive for Candida and four (2.7%) were positive for other yeasts. Among them, the first Indian C. dubliniensis isolate has been recovered. Molecular typing of selected C. albicans isolates by AP-PCR revealed two major genotypes based on the banding patterns. The susceptibilities of 30 Candida isolates to five antifungal agents including the new triazole voriconazole were determined in a micro-dilution test, according to the NCCLS protocol M 27. All the 22 C. albicans isolates were susceptible to five antimycotic agents (flucytosine, amphotericin B, fluconazole, voriconazole and itraconazole) except one isolate (VPCI-122), which was resistant to flucytosine (MIC > or = 64 mg l-1). The azole-resistant isolates reported here endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.     

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).     

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 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.     

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.     

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.     

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.     

Central nervous system infection due to Penicillium chrysogenum

Penicillium chrysogenum was isolated from three subsequent cerebrospinal fluid (CSF) specimens of a 73-year-old male patient without immunological compromise. The isolated was tested against five antifungal agents according to the NCCLS M38-P macrodilution method. MICs were determined as follows: amphotericin B (AMB), 2 microg ml(-1); fluconazole (FLZ), 8 microg ml(-1); itraconazole (ITZ), 1 microg ml(-1); flucytosine (5FC), 0.125 microg ml(-1); and terbinafine (TRB), 0.06 microg ml(-1). The patient has been cured with FLZ.     

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.