OBJECTIVE : This study aimed to explore the role of hormone replacement therapy (HRT) in susceptibility to vulvovaginal candidiasis (VVC) in a private vulval disease referral practice. METHODS : Between January 2009 and December 2010, 149 healthy, nondiabetic patients with vulvar conditions were compared for significant differences in vaginal swab result, age, and diagnosis between those using and not using HRT. Detailed clinical data were collected from those with VVC. RESULTS : The mean ages of the HRT (n = 70) and non-HRT (n = 79) groups were 62.5 and 62.5 years, respectively. Positive cultures for Candida were found in 34 (48.5%) of 70 patients on HRT and in 2 (3%) of 79 subjects not on HRT (p <.001). Culture-positive, clinical VVC was identified in 34 (49%) of 70 patients on HRT and in 1 (1%) of 79 patients not on HRT (p <.001). Candida species (32 Candida albicans and 2 Candida glabrata) were isolated from the 34 VVC patients, and of these, 23 (67%) had a history of recurrent or chronic candidiasis before menopause. All 34 had been previously treated with antifungal therapy without ceasing HRT and had been unresponsive to treatment or had relapse after treatment. In 27 (79%) of 34 patients, HRT was suspended during treatment. Of those who remained on HRT during treatment or resumed it after treatment, prophylactic antifungal treatment was initiated in 15 (44%) to prevent recurrence. All patients responded to the antifungal treatment provided HRT was suspended or prophylactic treatment was used. CONCLUSIONS : Postmenopausal women taking HRT are significantly more prone to develop VVC than women who are not and those with VVC are likely to have been susceptible to it before menopause.

Methods are described for extraction of DNA from the yeast form of Candida spp., followed by digestion and electrophoresis of DNA fragments. The resulting gel patterns (greater than 100 bands) were used to type Candida isolates. Four intense bands identified, three of which are present in each isolate (6 to 7, 3.7 or 4.2, and 2.5 to 3 kilobases), appear to be DNA encoding the rRNA. The methods proved to be both simple and reproducible. The patterns were shown to be stable through several hundred doublings from multiple single colonies. A survey of isolates showed that, on the basis of similarity of gel patterns, several Candida species could be sorted into mutually exclusive groups, and subgroups could be created. Analyses of this survey suggested the possible epidemiologic and taxonomic applications of these methods. DNA typing methods appear to offer important potential advantages over phenotyping methods. The methods provide a base for further epidemiologic studies and for further development of techniques, such as the use of cloned probes for studies of DNA homology.

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp.(n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.

We describe a patient with recurrent episodes of oropharyngeal candidiasis who required progressively higher doses of fluconazole to control and infection. The patient was treated for 14 infections over a 2-year period with doses of fluconazole that ranged from 100 to 800 mg per day. Clinical response, two methods of in vitro susceptibility testing, and molecular epidemiologic techniques were evaluated for 12 of the 14 episodes. Ultimately, the patient became unresponsive clinically to a dose of 800 mg of fluconazole per day. In vitro susceptibility testing of isolates obtained during these successive episodes of infection revealed the development of resistance to fluconazole, and molecular epidemiologic techniques confirmed the persistence of the same Candida albicans strain throughout all 12 episodes.

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans, including the ability to form germ tubes and chlamydospores. These similarities have caused significant problems in the identification of C. dubliniensis by the average clinical mycology laboratory. To facilitate the differentiation of these species, we investigated the growth of 120 isolates of C. dubliniensis and 98 C. albicans isolates at 42 and 45 degrees C on Emmons' modified Sabouraud glucose agar (SGA) and 10 isolates of each species in yeast-peptone-dextrose broth. None of the C. dubliniensis isolates grew on the agar or in the broth medium at 45 degrees C, while 11 isolates were capable of growing on SGA at 42 degrees C. In contrast, all of the C. albicans isolates but one grew at 45 degrees C on or in either medium. These reproducible results clearly demonstrate that the incubation of isolates suspected to be C. dubliniensis or C. albicans at 45 degrees C provides a simple, reliable, and inexpensive method for the differentiation of the two species.

Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.

Denture biofilms represent a protective reservoir for oral microbes. The study of the biology of Candida in these biofilms requires a reliable model. A reproducible model of C. albicans denture biofilm was developed and used to determine the susceptibility of two clinically relevant C. albicans isolates against 4 antifungals. C. albicans, growing as a biofilm, exhibited resistance to amphotericin B, nystatin, chlorhexidine, and fluconazole, with 50% reduction in metabolic activity (50% RMA) at concentrations of 8, 16, 128, and > 64 microg/mL, respectively. In contrast, planktonically cultured C. albicans were susceptible (50% RMA for the same antifungals was obtained at 0.25, 1.0, 4.0, and 0.5 microg/mL, respectively). In conclusion, results obtained by means of our biofilm model show that biofilm-associated C. albicans cells, compared with cells grown in planktonic form, are resistant to antifungals used to treat denture stomatitis.

When microbial strain-typing methods are compared, the most important characteristics are typeability, reproducibility, and discriminatory power. While typeability and reproducibility can be presented as numerical values, indices of discriminatory power have only recently been described. This paper examines the relationship between reproducibility and indices of discriminatory power. In an individual typing method, an inverse relationship between reproducibility and discriminatory power appears as the number of test differences required in order to distinguish between strains is increased. A method of standardizing the discriminatory power of a typing method to a predetermined reproducibility is presented. In this way the discriminatory powers of different typing methods can be compared while being standardized for the effect of reproducibility.

A system of 10 agar plate tests allowed presumptive identification of Candida albicans and differentiation of up to 512 strain types within the species. The yeast isolates were tested for acid and salt tolerance, proteinase production, resistance to 5-fluorocytosine and safranine, and assimilation of urea, sorbose, citrate and glycine. Media were inoculated semiquantitatively with a multiple-pronged device so that 55 yeasts and 5 reference strains could be printed on each plate at once. The results for the 9 strain differentiation tests were arranged in 3 groups to allow simple designation of types by 3-digit numbers. Mouth and vaginal samples from 85 patients and healthy volunteers yielded to 45 different strain types, of which types 153, 157 and 357 were the most prevalent. The reproducibility of the system was good, if attention was paid to the precision which the media were prepared and inoculated. The effect of test variables, including incubation temperature, medium pH and inoculum size, was assessed. Work is presently in progress to extend the system for presumptive identification of other clinically important Candida spp. and differentiation of their strain types.

Candida albicans strains were isolated from the oral cavities of 62 human immunodeficiency virus (HIV)-infected patients at different stages of HIV infection. Only patients with persistent generalized lymphadenopathy-acquired immunodeficiency syndrome (AIDS)-related complex or full-blown AIDS showed typical clinical symptoms for oral candidiasis. In general, the microbiological recovery of Candida strains from the oral cavity increased with more advanced stages of HIV infection. The antifungal activity of ketoconazole, itraconazole, nystatin, amphotericin B, and flucytosine against all 62 strains was evaluated by means of a photometer-read broth microdilution method for determination of the 30% inhibitory concentrations of the drugs. The 95% ranges of 30% inhibitory concentrations were as follows: less than or equal to 0.063 to 32 micrograms/ml for ketoconazole, less than or equal to 0.063 to 8 micrograms/ml for itraconazole, 0.5 to 4 micrograms/ml for nystatin, less than or equal to 0.063 to 4 micrograms/ml for amphotericin B, and less than or equal to 0.063 to 8 micrograms/ml for flucytosine. Two strains were resistant to flucytosine, one was resistant to ketoconazole, and three were resistant to itraconazole. Isolates from patients with full-blown AIDS showed significantly less susceptibility to itraconazole, amphotericin B, and flucytosine. Strains were biotyped by using the API 20C carbohydrate assimilation system. The major biotype accounted for 63.9% of the isolates. At repeated evaluation, a change in biotype pattern was seen in 27.3%.

The killer effect of 37 species of Candida, Cryptococcus, Hansenula, Pichia, Rhodotorula, Saccharomyces, and Trichosporon on 100 Candida albicans isolates of human and animal origin was studied. All of the C. albicans cultures were sensitive to one or more killer yeasts. The factors affecting the killer phenomenon on C. albicans were investigated for realizing a simple system for the differentiation of the 100 C. albicans isolates. By using this system, it was possible to differentiate up to 512 isolates of C. albicans according to their susceptibility to the killer effect of nine selected killer yeasts. The use of this method as an epidemiological marker in the case of presumptive nosocomial infections due to C. albicans is also reported.