Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy.

In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be > or = 6.25 micrograms/ml by the M27-P micro method and > or = 25 micrograms/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > or = itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole.     

Therapeutic options for the management of oropharyngeal and esophageal candidiasis in HIV/AIDS patients

Mucocutaneous candidiasis is frequently one of the first signs of HIV infection. Over 90% of patients with AIDS will develop oropharyngeal candidiasis at some time during their illness. Although numerous antifungal agents are available, azoles, both topical (clotrimazole) and systemic (fluconazole, itraconazole), have replaced older topical antifungals (gentian violet and nystatin) in the management of oropharyngeal candidiasis in these patients. The systemic azoles, itraconazole and fluconazole, are generally safe and effective agents in HIV-infected patients with oropharyngeal candidiasis. A concern in these patients is clinical relapse, which appears to be dependent on degree of immunosuppression and is more common following clotrimazole and ketoconazole than following fluconazole or itraconazole. Candida esophagitis is also of concern, since it occurs in more than 10% of patients with AIDS. Fluconazole is an integral part of the management of mucosal candidiasis. A cyclodextrin oral solution formulation of itraconazole has clinical response rates similar to fluconazole and is an effective alternative. In patients with fluconazole-refractory mucosal candidiasis, treatment options include itraconazole, amphotericin B oral suspension, and parenteral amphotericin B.     

Therapeutic Options for the Management of Oropharyngeal and Esophageal Candidiasis in HIV/AIDS Patients

Abstract

Mucocutaneous candidiasis is frequently one of the first signs of HIV infection. Over 90% of patients with AIDS will develop oropharyngeal candidiasis at some time during their illness. Although numerous antifungal agents are available, azoles, both topical (clotrimazole) and systemic (fluconazole, itraconazole), have replaced older topical antifungals (gentian violet and nystatin) in the management of oropharyngeal candidiasis in these patients. The systemic azoles, itraconazole and fluconazole, are generally safe and effective agents in HIV-infected patients with oropharyngeal candidiasis. A concern in these patients is clinical relapse, which appears to be dependent on degree of immunosuppression and is more common following clotrimazole and ketoconazole than following fluconazole or itraconazole. Candida esophagitis is also of concern, since it occurs in more than 10% of patients with AIDS. Fluconazole is an integral part of the management of mucosal candidiasis. A cyclodextrin oral solution formulation of itraconazole has clinical response rates similar to fluconazole and is an effective alternative. In patients with fluconazole-refractory mucosal candidiasis, treatment options include itraconazole, amphotericin B oral suspension, and parenteral amphotericin B.     

In vitro comparison of cilofungin alone and in combination with other antifungal agents against clinical isolates ofCandida species

Cilofungin, a lipopeptide antifungal agent, was tested for in vitro activity alone and in combination with ketoconazole, itraconazole, flucytosine and amphotericin B against 102 clinical isolates ofCandida species. At 48 hours all isolates ofCandida albicans, Candida tropicalis, Candida paratropicalis andCandida glabrata were inhibited by 5 mcg/ml of cilofungin. In contrast, the MIC90 forCandida krusei was 10 mcg/ml and forCandida parapsilosis >40 mcg/ml. The interaction of combinations of cilofungin with amphotericin B, itraconazole, ketoconazole and flucytosine was additive or indifferent at 48 hours for 100 %, 88 %, 78 % and 70 % of allCandida species isolates, respectively. Overall, cilofungin demonstrated good activity in vitro against mostCandida species isolates.     

Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole.

Ketoconazole and itraconazole were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Two isolates that had been previously identified as QC isolates for amphotericin B, fluconazole, and flucytosine (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were tested in accordance with the National Committee for Clinical Laboratory Standards M27-P guidelines. Each isolate was tested 20 times with the two antifungal agents in the five laboratories by using a lot of RPMI 1640 unique to each laboratory as well as a lot common to all five laboratories, thus generating 200 MICs per drug per organism. Overall, 96 to 99% of the MICs for each drug fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). By using these data, 3-log2 dilution QC ranges encompassing 98% of the observed MICs for three of the organism-drug combinations and 94% of the observed MICs for the fourth combination were established. These QC ranges are 0.064 to 0.25 micrograms/ml for both ketoconazole and itraconazole against C. parapsilosis ATCC 22019 and 0.125 to 0.5 micrograms/ml for both ketoconazole and itraconazole against C. krusei ATCC 6258.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Simplified bioassay method for measurement of flucytosine or ketoconazole.

A simple agar-well diffusion bioassay suitable for measurement of flucytosine or ketoconazole was developed by using Candida pseudotropicalis ATCC 46764 as the assay organism. A test medium composed of (per liter) 7 g of Trypticase peptone, 7 g of YNB (yeast-nitrogen base), 15 g of glucose, and 15 g of agar was seeded with an inoculum which had been grown to no. 2 McFarland turbidity after 4 to 6 h in YNB-glucose broth. Determinations of flucytosine or ketoconazole were performed without necessity of heating or diluting of serum samples to alleviate amphotericin B interference. A linear relationship between zone diameters and log10 concentration of the drugs was observed over the pharmacologically relevant ranges of 25 to 160 micrograms/ml for flucytosine and 0.5 to 20 micrograms/ml for ketoconazole. The mean coefficient of variability for samples measured on 5 separate days was 2.4% for flucytosin and 4.0% for ketoconazole. This assay represents a significant improvement over previous bioassay methods in that a single test system may be used for measurement of either flucytosine or ketoconazole, no serum dilution or pretreatment is required, inoculum preparation is accomplished entirely on the day of the assay, and sharp, clearly defined zones of inhibition are obtained with both drugs.     

First report of Cryptococcus laurentii meningitis and a fatal case of Cryptococcus albidus cryptococcaemia in AIDS patients.

We report the first case of Cryptococcus laurentii meningitis and a rare case of Cryptococcus albidus cryptococcaemia in AIDS patients. Both infections were treated with amphotericin B and flucytosine. The C. laurentii meningitis was controlled after 2 weeks of treatment with no evidence of infection 20 months later. The patient with C. albidus cryptococcaemia, despite the amphotericin B/flucytosine combination therapy, died on the 14th day of treatment. The minimum inhibitory concentrations (MICs) for C. laurentii, as determined by Etest on RPMI 1640 agar, were 0.25 microg ml(-1) of amphotericin B, 1.25 microg ml(-1) flucytosine, 4 microg ml(-1) fluconazole, 0.50 microg ml(-1) itraconazole and 1.0 microg ml(-1) of ketoconazole. The MIC of amphotericin B for C. albidus was 0.5 microg ml(-1), flucytosine 1.25 microg ml(-1), fluzonazole 4 microg ml(-1), itraconazole 0.5 microg ml(-1) and ketonazole 0.25 microg ml(-1). The agreement of the amphotericin B MIC values obtained in antibiotic medium 3 by the broth microdilution method, with those obtained on casitone medium by Etest, was within a two-dilution range for both isolates. C. laurentii may cause meningitis and may also involve the lungs in AIDS patients.     

In vitro activities of voriconazole and five licensed antifungal agents against Candida dubliniensis: comparison of CLSI M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods

We compared the in vitro activity of six antifungal agents against 62 isolates of Candida dubliniensis by the Clinical Laboratory Standards Institute (CLSI [formerly National Committee for the Clinical Laboratory Standards]) M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods and we studied the effect of the time of reading. For the azoles, voriconazole was the most potent in vitro followed by fluconazole, ketoconazole, and itraconazole. All the isolates were susceptible to amphotericin B and flucytosine. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible dose-dependent. At 24 hr, 100% of the isolates were susceptible to ketoconazole, amphotericin B, and flucytosine, 98% susceptible to voriconazole and fluconazole, and 95% for itraconazole. At 48 hr, 100% of the isolates remained susceptible for flucytosine and amphotericin B, 95% for voriconazole, 93% for fluconazole, 90% for ketoconazole, and 82% for itraconazole. The agreement between the CLSI and the other methods was better at 24 than 48 hr.     

Candida pseudorugosa sp. nov., a novel yeast species from sputum

Two yeast strains, strains XH 1026 and XH 1164, isolated from the sputum of an intensive care unit patient with acute pneumonia, were originally identified as Candida albicans and C. tropicalis, respectively. Sequence analysis of the 26S rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) region indicated that the two strains represent a novel yeast species closely related to C. rogusa. The name Candida pseudorugosa sp. nov. is therefore proposed (type strain, AS 2.3107 [CBS 10433]). The new species is able to grow at 42 degrees C and is resistant or insusceptible to amphotericin B (MIC, 2 microg/ml), caspofungin (MIC, 64 microg/ml), itraconazole (MIC, 1 microg/ml), and nystatin (MIC, 16 microg/ml); dose-dependent susceptible to fluconazole (MIC, 16 microg/ml); and susceptible to flucytosine (MIC, 0.125 microg/ml) and voriconazole (MIC, 0.125 to 0.25 microg/ml). The code for C. pseudorugosa sp. nov. provided by the API 20C AUX system is identical to that for C. rugosa. The colonies of the new species on CHROMagar Candida appear blue-green, similar to those of C. albicans. In addition to the molecular method based on D1/D2 domain or ITS region sequencing, use of the combination of the API system and CHROMagar Candida is helpful for the correct identification of C. pseudorugosa sp. nov.     

In vitro antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method.

Fungal infections have dramatically increased in recent years, along with the increase of drug-resistant isolates in immunocompromised patients. Ninety eight Candida species obtained from blood cultures at the Tri-Service General Hospital, Taiwan, from 1998 to 2000 were studied. These included 50 Candida albicans, 13 Candida glabrata, 24 Candida tropicalis and 11 Candida parapsilosis isolates. To investigate their susceptibility to commonly used antifungal drugs, minimum inhibitory concentrations (MIC) of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined. Both the National Committee for Clinical Laboratory Standards reference broth macrodilution method and E-test were used in parallel. Ninety five isolates (95/98, 96.94%) were susceptible to amphotericin B at a concentration < or = 1 microg/mL. All isolates (100%, 98/98) were susceptible to flucytosine. Approximately 30% of these Candida isolates were resistant to fluconazole. The MIC for 90% of isolates (MIC90) values for both methods for these isolates were 0.5 microg/mL for amphotericin B, 32 microg/mL for fluconazole, 0.25 microg/mL for flucytosine (0.125 microg/mL by E-test method), and 4 microg/mL for ketoconazole. MIC for 50% of isolates (MIC50) values for these agents were 0.25, 2, 0.06, and 0.06 microg/mL, respectively. The essential agreement of MIC values within 2 dilutions for the 2 methods was 99.0% for amphotericin B, 90.8% for ketoconazole, 92.9% for fluconazole, and 91.8% for flucytosine. This study showed that E-test has equivalent performance to the broth macrodilution method and can be used as an alternative MIC technique for antifungal susceptibility testing.     

Comparative evaluation of PASCO and national committee for clinical laboratory standards M27-A broth microdilution methods for antifungal drug susceptibility testing of yeasts

The PASCO antifungal susceptibility test system, developed in collaboration with a commercial company, is a broth microdilution assay which is faster and easier to use than the reference broth microdilution test performed according to the National Committee for Clinical Laboratory Standards (NCCLS) document M27-A guidelines. Advantages of the PASCO system include the system's inclusion of quality-controlled, premade antifungal panels containing 10, twofold serial dilutions of drugs and a one-step inoculation system whereby all wells are simultaneously inoculated in a single step. For the prototype panel, we chose eight antifungal agents for in vitro testing (amphotericin B, flucytosine, fluconazole, ketoconazole, itraconazole, clotrimazole, miconazole, and terconazole) and compared the results with those of the NCCLS method for testing 74 yeast isolates (14 Candida albicans, 10 Candida glabrata, 10 Candida tropicalis, 10 Candida krusei, 10 Candida dubliniensis, 10 Candida parapsilosis, and 10 Cryptococcus neoformans isolates). The overall agreements between the methods were 91% for fluconazole, 89% for amphotericin B and ketoconazole, 85% for itraconazole, 80% for flucytosine, 77% for terconazole, 66% for miconazole, and 53% for clotrimazole. In contrast to the M27-A reference method, the PASCO method classified as resistant seven itraconazole-susceptible isolates (9%), two fluconazole-susceptible isolates (3%), and three flucytosine-susceptible isolates (4%), representing 12 major errors. In addition, it classified two fluconazole-resistant isolates (3%) and one flucytosine-resistant isolate (1%) as susceptible, representing three very major errors. Overall, the agreement between the methods was greater than or equal to 80% for four of the seven species tested (C. dubliniensis, C. glabrata, C. krusei, and C. neoformans). The lowest agreement between methods was observed for miconazole and clotrimazole and for C. krusei isolates tested against terconazole. When the data for miconazole and clotrimazole were removed from the analysis, agreement was >/=80% for all seven species tested. Therefore, the PASCO method is a suitable alternative procedure for the testing of the antifungal susceptibilities of the medically important Candida spp. and C. neoformans against a range of antifungal agents with the exceptions only of miconazole and clotrimazole and of terconazole against C. krusei isolates.     

Comparative study of broth macrodilution and microdilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards' proposed standard.

A comparative study of broth macro- and microdilution methods for susceptibility testing of fluconazole, itraconazole, flucytosine, and amphotericin B was conducted with 273 yeasts. The clinical isolates included 100 Candida albicans, 28 Candida tropicalis, 25 Candida parapsilosis, 15 Candida lusitaniae, 15 Candida krusei, 50 Cryptococcus neoformans var. neoformans, 25 Torulopsis (Candida) glabrata, and 15 Trichosporon beigelii strains. Both methods were performed according to the National Committee for Clinical Laboratory Standards' (NCCLS) recommendations (document M27-P). For fluconazole, itraconazole, and flucytosine, the endpoint was the tube that showed 80% growth inhibition compared with the growth control for the macrodilution method and the well with slightly hazy turbidity (score 1) compared with the growth control for the microdilution method. For amphotericin B, the endpoint was the tube and/or well in which there was absence of growth. For the reference macrodilution method, the MICs were determined after 48 h of incubation for Candida spp., T. glabrata, and T. beigelii and after 72 h for C. neoformans var. neoformans. For the microdilution method, either the first-day MICs (24 h for all isolates other than C. neoformans and 48 h for C. neoformans var. neoformans) or the second-day MICs (48 and 72 h, respectively) were evaluated. The agreement within one doubling dilution of the macrodilution reference for all drugs was higher with the second-day MICs than with the first-day MICs for the microdilution test for most of the tested strains. General agreement was 92% for fluconazole, 85.7% for itraconazole, 98.3% for flucytosine, and 96.4% for amphotericin B. For C. neoformans var. neoformans and T. beigelii, the agreement of the first-day reading was higher than that of the second-day reading for fluconazole (94 versus 92%, respectively, for C. neoformans var. neoformans, and 86.7 versus 80%, respectively, for T. beigelii). Our studies indicate that the microdilution technique performed following the NCCLS guidelines with a second-day reading is a valid alternative method for testing fluconazole, itraconazole, flucytosine, and amphotericin B against these eight species of yeasts.     

Nosocomial outbreak caused by Scedosporium prolificans (inflatum): four fatal cases in leukemic patients.

Four cases of fatal disseminated Scedosporium prolificans (inflatum) infection occurring in neutropenic patients are reported. Because of hospital renovation, the patients were cared for in a temporary hematologic facility. S. prolificans (inflatum) was isolated from blood cultures of these four patients, two of whom underwent full necropsy, and revealed abundant vegetative hyphae and ovoid conida with truncate bases in many organs. In vitro susceptibility testing of fungal strains showed all isolates to be resistant to amphotericin B, flucytosine, miconazole, ketoconazole, fluconazole, and itraconazole, with MICs greater than 16 micrograms/ml. The reported infections, two in each of two rooms, occurred over a period of 1 month, with very similar clinical outcomes. Circumstancial evidence suggested a nosocomial outbreak, but the environmental samples collected from the rooms, corridors, and adjacent areas did not yield S. prolificans (inflatum). Nevertheless, circumstantial evidence suggested a nosocomial outbreak of S. prolificans (inflatum) infection.     

Isolation and characterization of a polyene-resistant variant of Candida tropicalis.

An atypical variant of Candida tropicalis was recovered from multiple specimens from a patient who had been a recipient of a bone marrow transplant. This yeast variant showed atypical morphology on corn meal agar distinguishable from typical isolates of C. tropicalis by the production of clusters of blastospores. Isolates of the variant produced acid, but no gas, from maltose and sucrose in fermentation tests. Isolates from blood, pleural fluid, respiratory secretions, and stool specimens were susceptible to amphotericin B and nystatin in an agar dilution system. However, eight isolates of the variant C. tropicalis recovered over a period of 4 weeks from the patient's urine after amphotericin B therapy were found to be resistant to amphotericin B and nystatin. The isolate recovered after 7 days of therapy had minimal inhibitory concentrations of 100 micrograms of amphotericin B and 20 micrograms of nystatin per ml, whereas the seven isolates recovered subsequently had minimal inhibitory concentrations of greater than 500 micrograms of amphotericin B and 50 micrograms of nystatin per ml. The resistant isolates concomitantly lost the capacity to utilize amino acids that susceptible isolates could utilize. Ultraviolet absorption spectra of nonsaponifiable fractions of whole cells showed that resistant isolates lacked ergosterol, which susceptible isolates contained.     

In vitro sensitivity of Aspergillus to terbinafine: comparative study on amphotericin B, 5-fluorocytosine and ketoconazole

Terbinafine, a new antifungal agent derived from allylamine, effective by oral route, was tested against 32 different strains of Aspergilli to determine its in vitro efficiency compared with amphotericin B, 5-fluorocytosine and ketoconazole. Antifungal sensitivity was determine by the MIC method in liquid and solid mediums (Sabouraud, Casitone, YMA, YMB, YNB): with MIC between 0.005 and 5 micrograms/ml, whatever the species and the techniques, terbinafine is more efficient than amphotericin B, ketoconazole (0.5 to 100 micrograms/ml) and 5-FC (1-greater than 100 micrograms/ml).     

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.     

Flucytosine Primary Resistance in Candida Species and Cryptococcus neoformans

 The in vitro activity of flucytosine (5FC) against 1,140 clinical isolates of Candida spp. and Cryptococcus neoformans was evaluated and compared with the activity of amphotericin B, fluconazole and itraconazole. Overall, 87.72% (1,000/1,140) of yeasts were susceptible to 5FC. This agent showed less potent in vitro activity against Candida glabrata, Candida krusei, Candida guilliermondii and Cryptococcus neoformans (MIC90s, 8–16 μg/ml) and intermediate activity or resistance to 6.5% of Candida albicans, 5.1% of Candida tropicalis and 0.8% of Candida parapsilosis strains. Amphotericin B showed potent activity against isolates with an MIC of 5FC≥8 μg/ml. A total of 112 of 140 strains that were 5FC-intermediate or -resistant showed decreased susceptibility to azoles (P<0.01).     

Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases

There are limited data regarding the antifungal susceptibility of yeast causing vulvovaginal candidiasis, since cultures are rarely performed. Susceptibility testing was performed on vaginal yeast isolates collected from January 1998 to March 2001 from 429 patients with suspected vulvovaginal candidiasis. The charts of 84 patients with multiple positive cultures were reviewed. The 593 yeast isolates were Candida albicans (n = 420), Candida glabrata (n = 112), Candida parapsilosis (n = 30), Candida krusei (n = 12), Saccharomyces cerevisiae ( n = 9), Candida tropicalis (n = 8), Candida lusitaniae (n = 1), and Trichosporon sp. (n = 1). Multiple species suggesting mixed infection were isolated from 27 cultures. Resistance to fluconazole and flucytosine was observed infrequently (3.7% and 3.0%); 16.2% of isolates were resistant to itraconazole (MIC > or = 1 microg/ml). The four imidazoles (econazole, clotrimazole, miconazole, and ketoconazole) were active: 94.3 to 98.5% were susceptible at < or =1 microg/ml. Among different species, elevated fluconazole MICs (> or = 16 microg/ml) were only observed in C. glabrata (15.2% resistant [R], 51.8% susceptible-dose dependent [S-DD]), C. parapsilosis (3.3% S-DD), S. cerevisiae (11.1% S-DD), and C. krusei (50% S-DD, 41.7% R, considered intrinsically fluconazole resistant). Resistance to itraconazole was observed among C. glabrata (74.1%), C. krusei (58.3%), S. cerevisiae (55.6%), and C. parapsilosis (3.4%). Among 84 patients with recurrent episodes, non-albicans species were more common (42% versus 20%). A > or = 4-fold rise in fluconazole MIC was observed in only one patient with C. parapsilosis. These results support the use of azoles for empirical therapy of uncomplicated candidal vulvovaginitis. Recurrent episodes are more often caused by non-albicans species, for which azole agents are less likely to be effective.     

Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp

The influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candida spp. clinical isolates (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida lusitaniae [10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (P < 0.01). A longer exponential phase and shorter lag phase were also observed. The A540 values at 24 h of incubation with medium containing glucose and an inoculum of 10(5) CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis (A540 = 0.26 +/- 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10(5) CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10(5) CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.