Micafungin enhances neutrophil fungicidal functions against Candida pseudohyphae

We evaluated the effect of the combination of micafungin and polymorphonuclear leukocytes (PMN) against hyphae of Candida albicans and Candida dubliniensis. Micafungin enhanced the PMN oxidative burst dose dependently. The combination was synergistic (C. albicans) or additive (C. dubliniensis); when PMN were pretreated with granulocyte-macrophage colony-stimulating factor, the combination was more effective.     

Echinocandin susceptibility testing of Candida spp. Using EUCAST EDef 7.1 and CLSI M27-A3 standard procedures: analysis of the influence of bovine serum albumin supplementation, storage time, and drug lots

The MICs of echinocandins against Candida isolates with fks mutations are higher than those for wild-type (WT) isolates. However, the MIC ranges for susceptible and mutant populations overlap or are poorly separated. It was recently reported that a greater separation could be achieved in the presence of serum. To more fully explore this possibility, we compared the performances of the reference microdilution methods by using standard and bovine serum albumin (BSA)-supplemented growth medium. Anidulafungin, caspofungin, and micafungin MICs were determined according to EUCAST and CLSI methods and with 50% BSA in the medium for 93 clinical isolates, including Candida albicans (20/10 [number of isolates/number of mutants]), C. glabrata (19/10), C. dubliniensis (2/1), C. krusei (16/3), C. parapsilosis (19), and C. tropicalis (19/4) isolates. Stability of the plates was tested after storage at -80°C for 2 and 6 months, and the performance of two different lots of caspofungin was investigated. The addition of BSA to the medium resulted in higher MICs (1 to 9 2-fold dilution steps) for all isolates and compounds. The increases were greatest for anidulafungin and micafungin and, among WT isolates, for C. parapsilosis. The number of very major errors (VMEs) was reduced (24% [20/84 isolates] versus ≤ 7% [6/84 isolates]) using BSA-supplemented EUCAST medium but not using BSA-supplemented CLSI medium (6% versus 9%). MIC results were unchanged after 6 months of storage of test plates. The two lots of caspofungin yielded identical results. Addition of BSA to the EUCAST medium increases the ability to differentiate between WT isolates and isolates harboring resistance mutations.     

Patterns of amphotericin B killing kinetics against seven Candida species

In a previous study tolerance to amphotericin B (AMB) was found among Candida parapsilosis and C. dubliniensis strains by seeding the whole volumes of wells used for MIC determinations, and minimum fungicidal concentrations (MFC) for non-C. albicans Candida strains were demonstrated to be above the levels safely achievable in serum. As an extension of that study, we performed time-kill assays with 26 blood culture isolates (6 C. albicans, 5 C. parapsilosis, 5 C. krusei, 4 C. glabrata, 3 C. lusitaniae, and 3 C. tropicalis isolates), 3 oropharyngeal C. dubliniensis isolates, 3 AMB-susceptible isolates (ATCC 90028, ATCC 22019, ATCC 6254), and 6 AMB-resistant isolates (ATCC 200955, ATCC 200956, ATCC 200950, ATCC 200951, ATCC 200952, ATCC 200953) using RPMI 1640 medium and 0.12 to 32 microg of AMB per ml and determined the numbers of CFU per milliliter at 0, 2, 4, 8, 12, 24, and 48 h. MFCs and time-kill patterns were species specific (MFCs, < or =1 microg/ml for all C. dubliniensis and C. albicans isolates except AMB-resistant strain ATCC 200955; MFCs, 2 to >16 microg/ml for the other isolates). The times required to reach the fungicidal endpoint (99.9% killing) at four times the MIC were 2 h for C. albicans and C. dubliniensis, 16 h for C. glabrata, 24 h for C. parapsilosis and C. lusitaniae, and > or =40 h for C. tropicalis and C. krusei. The killing rate increased as the AMB concentration was increased up to 2 microg/ml. The highest killing rates were achieved for C. albicans, C. dubliniensis, and C. lusitaniae, while viable C. tropicalis, C. krusei, and C. parapsilosis cells were present after 48 h (MICs, < or =2 microg/ml) when AMB was used at 2 microg/ml. Time-kill curves and MFCs can detect viable cells after 48 h when AMB is used at > or =2 microg/ml. The failure of AMB treatment could be due to its poor killing activity against some species at the concentrations reached in patients' serum.     

Application of hypertonic Sabouraud glucose agar for differentiation of Candida dubliniensis from Candida albicans

An evaluation of hypertonic Sabouraud glucose agar (SGA) with 6.5% NaCl for phenotypic differentiation of Candida dubliniensis from Candida albicans is reported. Identity of the test fungi (C. albicans, 84; C. dubliniensis, 18) was based on their typical phenotypic characteristics and confirmed by a diagnostic polymerase chain reaction that targets the novel C. dubliniensis group I intron in the large ribosomal subunit. At 96 h of incubation at 28 °C, all of the 84 C. albicans isolates showed growth on hypertonic SGA contrary to the consistently negative results with the 20 C. dubliniensis isolates. In strong contrast, chlamydospore formation on Staib agar yielded 10 (11.9%) false-positive results and 74 (88%) of the test C. albicans isolates showed false-negative results at 45 °C. We conclude that hypertonic SGA with 6.5% NaCl can be recommended for wider application as a reliable and inexpensive medium for routine differentiation of C. dubliniensis from C. albicans.     

A simple xylose-based agar medium for the differentiation of Candida dubliniensis and Candida albicans

The utility of xylose-based agar medium for differentiation of Candida dubliniensis from Candida albicans is evaluated. All C. dubliniensis isolates failed to grow on this medium, while C. albicans isolates yielded good growth. This simple in-house medium offers an inexpensive alternative to commercial yeast identification systems for resource poor settings.     

Evaluation of Bichro-Dubli Fumouze to distinguish Candida dubliniensis from Candida albicans

We have evaluated the ability of the Bichro-Dubli Fumouze (Fumouze Diagnostics, Levallois-Perret, France) latex agglutination test to identify colonies of Candida dubliniensis grown on different media. The test was positive for 103 of 106 isolates of C. dubliniensis and negative for Candida albicans and other Candida species studied. The sensitivity and specificity of the test were 97.1% and 100%, respectively. The test is very rapid, simple, and reliable giving the same results independently of whether the colonies are grown previously on Sabouraud dextrose agar, CHROMagar Candida medium, Candida ID2 medium, or CHROMagar-Pal's medium.     

In vitro activity of micafungin (FK-463) against Candida spp.: microdilution,time-kill,and postantifungal-effect studies

We evaluated the in vitro activity of the new echinocandin antifungal micafungin against Candida spp. using microdilution and time-kill methods. Additionally, we examined the postantifungal effect (PAFE) of micafungin. Finally, we evaluated the effect of the addition of serum and plasma on the MIC of micafungin. Four Candida albicans isolates and two isolates of each Candida glabrata, Candida krusei, and Candida tropicalis were selected for testing. The MICs of micafungin were determined in RPMI 1640 medium buffered with morpholinepropanesulfonic acid alone and with the addition of 10, 20, and 50% human serum and plasma. MICs were determined by using two endpoints: a prominent reduction in growth (the MIC at which 80% of isolates are inhibited [MIC(80)]) and complete visual inhibition of growth (MIC(100)). The minimum fungicidal concentration (MFC) of micafungin for each isolate was also determined. Time-kill curves were determined for each isolate in RPMI 1640 medium with micafungin at concentrations ranging from 0.125 to 16 times the MIC(80) to assess the correlation between MIC(80) and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC(80). The MIC(80)s for the test isolates ranged from 0.0039 to 0.25 micro g/ml. Overall, the addition of serum or plasma increased the MIC 6 to 7 doubling dilutions for C. albicans and 3 to 4 doubling dilutions for C. krusei and C. tropicalis. Micafungin time-kill studies demonstrated fungicidal activity at concentrations ranging from 4 to 16 times the MIC(80). Micafungin is very potent agent against a variety of Candida spp., producing fungicidal activity against 7 of 10 isolates tested. A PAFE was observed against all isolates. The PAFE was influenced by the drug concentration, with the highest concentration resulting in the longest observed PAFE in each case. The highest concentration tested, four times the MIC, resulted in a PAFE of more than 9.8 h for 5 of 10 isolates tested (range, 0.9 to > or =20.1 h).     

Incubation at room temperature may be an independent factor that induces chlamydospore production in Candida dubliniensis

Production of chlamydospores is one of the phenotypic features used to differentiate Candida albicans and Candida dubliniensis. C. albicans produces few chlamydospores on only cornmeal/rice-Tween agar at room temperature, whereas C. dubliniensis produces abundant chlamydospores at this temperature both on cornmeal agar and some other commonly used media. We tried to determine whether the room temperature is the main factor that induces chlamydospore production of C. dubliniensis, regardless of the medium used. For this purpose, 100 C. albicans and 24 C. dubliniensis isolates were tested for chlamydospore production at room temperature and at 37 degrees C on some routinely used media, including eosin-methylene blue agar (EMB), nutrient agar (NA), nutrient broth (NB), and also on an investigational medium, phenol red agar (PR). At 37 degrees C, none of the isolates produced chlamydospores on any of the tested media. At 26 degrees C, all C. dubliniensis isolates produced abundant chlamydospores and pseudohyphae after 24-48 h on all tested media. At this incubation temperature, all C. albicans isolates failed to produce chlamydospores and pseudohyphae on EMB, NA, and NB, whereas 2 of the C. albicans isolates produced a few chlamydospores on PR. We also observed that all C. dubliniensis isolates tested on EMB and PR produced rough colonies with a hyphal fringe around the colonies, whereas none of the C. albicans isolates showed this property. In conclusion, incubation at 26 degrees C may play the key role for production of abundant chlamydospores and pseudohyphae by C. dubliniensis. Comprehensive molecular studies are needed to clarify the genetic basis of this observation. Using EMB and PR may be an inexpensive, a time-saving, and a simple way of presumptive identification of C. dubliniensis based on chlamydospore formation and colony morphology.     

Evaluation of 5 new media containing extracts of seeds applied to Candida dubliniensis screening

Candida dubliniensis is a recently described pathogenic species that shares many phenotypic features with Candida albicans and so may be misidentified in microbiologic laboratories. The aim of this study is to find a useful and cost-effective method suitable for screening C. dubliniensis before proceeding to further identification. We examined the colony morphology and chlamydospore production of 26 C. dubliniensis isolates and 100 C. albicans isolates on the following 5 proposed media: sesame seed agar (SSA), rapeseed agar, canary grass seed agar, millet seed agar, and linseed agar (LA). The best results were obtained with SSA and LA because all 26 C. dubliniensis isolates showed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 h of incubation at 25 degrees C. All C. albicans isolates (100%) showed smooth colonies without hyphal fringes or chlamydospores. These 2 media consist of new and simple tools for presumptive differentiation of C. dubliniensis from C. albicans.     

The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory

Candida dubliniensis is a recently described pathogenic yeast that is closely related to C. albicans. The germ tube test is used routinely in diagnostic laboratories for the identification of C. albicans, and C. dubliniensis may also produce germ tubes under the same conditions. We evaluated a previously described method for differentiating between the two species using Niger seed agar (Staib agar). The aim was to find a useful, user-friendly and cost-effective method for use in diagnostic work. C. albicans produces only yeast cells on this medium after 24 h at 37 degrees C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed. Of 495 yeasts isolated in, or sent for identification to, a diagnostic mycology laboratory 9 isolates (1.8%) were found to be C. dubliniensis. The method was found to be valuable for screening yeasts before proceeding to further identification if positive for hyphal/pseudohyphal growth on Niger seed agar. This method is therefore suitable for the screening of selected yeast isolates in order to identity C. dubliniensis and will further our understanding of the clinical importance of this species.     

In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates

Caspofungin is an echinocandin antifungal agent with broad-spectrum activity against Candida and Aspergillus spp. The in vitro activities of caspofungin against 3,959 isolates of Candida spp. obtained from over 95 different medical centers worldwide were compared with those of fluconazole and itraconazole. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the NCCLS method using RPMI 1640 as the test medium. Caspofungin was very active against Candida spp. (MIC at which 90% of the isolates were inhibited [MIC(90)], 1 micro g/ml; 96% of MICs were < or =2 micro g/ml). Candida albicans, C. dubliniensis, C. tropicalis, and C. glabrata were the most susceptible species of Candida (MIC(90), 0.25 to 0.5 micro g/ml), and C. guilliermondii was the least susceptible (MIC(90), >8 micro g/ml). Caspofungin was very active against Candida spp., exhibiting high-level resistance to fluconazole and itraconazole (99% of MICs were < or =1 micro g/ml). These results provide further evidence for the spectrum and potency of caspofungin activity against a large and geographically diverse collection of clinically important isolates of Candida spp.     

Multicenter prospective surveillance of oral Candida dubliniensis among adult Brazilian human immunodeficiency virus-positive and AIDS patients.

The incidence of C. dubliniensis in South America has not yet been determined. In the present study, oral swab samples were taken from 108 HIV-infected/AIDS individuals attending 6 separate Brazilian HIV-treatment centers to determine the incidence of C. dubliniensis in this population. Swabs were plated onto CHROMagar Candida medium and 155 isolates, presumptively identified as C. albicans or C. dubliniensis were further investigated. In a preliminary screen for C. dubliniensis, 13 of the 155 isolates showed no or poor growth at 42 degrees C, and all them were subjected to randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) analysis using C. dubliniensis-specific primers. We confirmed that 4 out of 13 isolates were C. dubliniensis, representing an incidence rate of 2.8% for the Brazilian HIV-infected population infected with yeasts exhibiting green colonies on CHROMagar Candida. This value is significantly lower than those reported in Ireland and the United States.     

Candida albicans and Candida dubliniensis respond differently to echinocandin antifungal agents in vitro

Candida dubliniensis isolates tested for susceptibility to anidulafungin, caspofungin, and micafungin commonly showed artifactual regrowth and/or trailing effects with MIC tests done under conditions involving a high initial yeast concentration. The artifacts were less common with Candida albicans and seldom seen for either species under Clinical and Laboratory Standards Institute method M27-A test conditions.     

Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild-type and fks hot spot mutant Candida isolates

Disk diffusion testing has recently been standardized by the CLSI, and susceptibility breakpoints have been established for several antifungal compounds. For caspofungin, 5-μg disks are approved, and for micafungin, 10-μg disks are under evaluation. We evaluated the performances of caspofungin and micafungin disk testing using a panel of Candida isolates with and without known FKS echinocandin resistance mechanisms. Disk diffusion and microdilution assays were performed strictly according to CLSI documents M44-A2 and M27-A3. Eighty-nine clinical Candida isolates were included: Candida albicans (20 isolates/10 mutants), C. glabrata (19 isolates/10 mutants), C. dubliniensis (2 isolates/1 mutant), C. krusei (16 isolates/3 mutants), C. parapsilosis (14 isolates/0 mutants), and C. tropicalis (18 isolates/4 mutants). Quality control strains were C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. The correlations between zone diameters and MIC results were good for both compounds, with identical susceptibility classifications for 93.3% of the isolates by applying the current CLSI breakpoints. However, the numbers of fks hot spot mutant isolates misclassified as being susceptible (S) (very major errors [VMEs]) were high (61% for caspofungin [S, ≥11 mm] and 93% for micafungin [S, ≥14 mm]). Changing the disk diffusion breakpoint to S at ≥22 mm significantly improved the discrimination. For caspofungin, 1 VME was detected (a C. tropicalis isolate with an F76S substitution) (3.5%), and for micafungin, 10 VMEs were detected, the majority of which were for C. glabrata (8/10). The broadest separation between zone diameter ranges for wild-type (WT) and mutant isolates was seen for caspofungin (6 to 12 mm versus -4 to 7 mm). In conclusion, caspofungin disk diffusion testing with a modified breakpoint led to excellent separation between WT and mutant isolates for all Candida species.     

Species-specific differences in the susceptibilities of biofilms formed by Candida bloodstream isolates to echinocandin antifungals

The echinocandin susceptibilities of bloodstream Candida isolates growing in a biofilm was investigated. Within the therapeutic range of concentrations of each drug, caspofungin and micafungin were active against biofilms formed by Candida albicans or C. glabrata but not those formed by C. tropicalis or C. parapsilosis.     

National surveillance of species distribution in blood isolates of Candida species in Japan and their susceptibility to six antifungal agents including voriconazole and micafungin

OBJECTIVES: The aim of this study was to evaluate species distribution and antifungal susceptibility of Candida blood isolates in Japan. METHODS: In a 1 year surveillance programme, 535 Candida blood isolates were collected. Identification of species was followed by examination with the broth microdilution method, as described in NCCLS M27-A2, of antifungal susceptibility to six agents, including voriconazole and micafungin, with readings after 24 and 48 h of incubation. RESULTS: The overall species distribution was: 41% Candida albicans, 23% Candida parapsilosis, 18% Candida glabrata, 12% Candida tropicalis and 2% Candida krusei. The concentrations of fluconazole necessary to inhibit 90% of the isolates (MIC(90)) at 24/48 h were 0.25/1 mg/L for C. albicans, 0.5/2 mg/L for C. parapsilosis, 4/32 mg/L for C. glabrata and 4/>128 mg/L for C. tropicalis. Percentages of fluconazole resistance were 1.8% for C. albicans, 0.8% for C. parapsilosis, 5.2% for C. glabrata and 3.2% for C. tropicalis, taking the tendency of trailing growth of C. tropicalis into account. MIC(90) of voriconazole was 0.5 mg/L, although 35% of isolates less susceptible (>/=16 mg/L) to fluconazole showed resistance (>/=2 mg/L). Micafungin was very active against all species (MIC(90), 0.03 mg/L) except for C. parapsilosis (MIC(90), 2 mg/L). CONCLUSIONS: These data suggest that, in Japan, the species distribution of Candida bloodstream infections and the fluconazole resistance rate are similar to those reported previously in North America and Europe. Voriconazole and micafungin appear to have strong in vitro activity against Candida blood isolates, although continuing surveillance and further clinical research are needed.     

Differentiation of Candida dubliniensis from Candida albicans on rosemary extract agar and oregano extract agar

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans and therefore, may be misidentified in microbiological laboratories. Because molecular methods can be onerous and unfeasible in routine mycological laboratories with restricted budgets such as those in developing countries, phenotypic techniques have been encouraged in the development of differential media for the presumptive identification of these species. We examined the colony morphology and chlamydospore production of 30 C. dubliniensis isolates and 100 C. albicans isolates on two new proposed media: rosemary (Rosmarinus officinalis) extract agar (REA) and oregano (Origanum vulgare) extract agar (OEA). These substrates are traditionally used as spices and medicinal herbs. In both of these media, all C. dubliniensis isolates (100%) showed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 hr of incubation at 25 degrees C. In contrast, under the same conditions, all isolates of C. albicans (100%) showed smooth colonies without hyphal fringes or chlamydospores. In conclusion, REA and OEA offer a simple, rapid, and inexpensive screening media for the differentiation of C. albicans and C. dubliniensis.     

Progressive loss of echinocandin activity following prolonged use for treatment of Candida albicans oesophagitis

OBJECTIVES: To illustrate the progressive loss of cross-echinocandin activity on Candida albicans isolates with strong clonal homology from a patient with advanced HIV infection and chronic oesophagitis progressively resistant to uninterrupted micafungin treatment. METHODS: Antifungal susceptibility profiles for different antifungal agents were determined against serial C. albicans isolates retrieved before and during therapy. Multilocus sequencing typing (MLST) was performed on each of the isolates. FKS1 mutations conferring reduced susceptibility to echinocandin drugs were determined by DNA sequence analysis. RESULTS: Four C. albicans isolates showing identical allelic homology were retrieved from the patient at the initiation and during therapy with micafungin. The progressive lack of clinical response to micafungin therapy was associated with increased MICs of all three echinocandin drugs (caspofungin, micafungin and anidulafungin) in association with the acquisition of mutations in the FKS1 gene. CONCLUSIONS: This report documents for the first time a progressive loss of activity of all three echinocandin drugs against clonally related C. albicans isolates following long-term clinical exposure to this new class of antifungal agents.     

New PCR primer pairs specific for Candida dubliniensis and detection of the fungi from the Candida albicans clinical isolates in Japan

The genetic patterns of Candida dubliniensis, including type strain, were analyzed by random amplified polymorphic DNA (RAPD) method in comparison with those of reference Candida albicans genotypes A, B and C strains. This RAPD pattern analysis showed that the patterns of two strains of C. dubliniensis including the type strain are very similar to the three oligo PCR primers used, but different from other C. albicans genotypes A, B and C strains with most of the PCR primers tested. The RAPD band considered to be specific for C. dubliniensis in one oligo primer was extracted and sequenced. Based on the sequence information (417-bp), two new PCR primer pairs specific for C. dubliniensis were prepared. The survey of 58 strains of Candida clinical isolates using these newly prepared PCR primer pairs identified as C. albicans in Japan suggested the presence of one strain of C. dubliniensis. The phenotypic characteristics and the sequence analysis of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA of the strain confirmed that it belongs to C. dubliniensis. The usefulness and specificity of these PCR primer pairs for the identification of C. dubliniensis are discussed.     

Comparison of Albicans ID2 agar plate with the germ tube for presumptive identification of Candida albicans

Albicans ID2 (bioMérieux, France) is a commercially available chromogenic medium that allows rapid and specific macroscopic identification of Candida albicans and facilitates the differentiation of species in mixed cultures. We compared it with the standard method for the identification of yeast species, the germ tube test (GT). This study involved 423 clinical isolates, including 163 C. albicans and 260 non-albicans yeasts. Sensitivity of Albicans ID2 agar plates regarding the identification of C. albicans were 98.2% after 48 h of incubation and specificity of 96.6%. This method using rapid enzymatic method shows the same similar sensitivity than the GT test The false negative rate (1.8%) for the GT test is consistent with that previously reported. None tests discriminated between C. albicans and C. dubliniensis isolates.