Oral candidosis is a common opportunistic infection in elderly denture wearers. For instance, 60% of denture wearers over the age of 60 years in Denmark suffer from Candida-associated denture stomatitis. Although these superficial infections are the most prevalent form of the disease, systemic candidal infections are increasingly seen particularly in the compromised, such as those with AIDS.

Acrylic dentures are an important predisposing factor for oral candidosis as these appliances, which are usually ill fitting and with suboptimal hygiene, act as reservoirs of infection. For instance, high salivary yeast counts are much more common in full-denture wearers than in dentate individuals, and yeasts are demonstrable in 78–100% of patients with denture-induced stomatitis as contrasted with 30–60% of the non-denture-wearing general population. It is thought that the ability of Candida spp., especially C. albicans, to adhere to acrylic surfaces may be important in the pathogenesis of the disease as adherence is apparently the initial step in microbial colonization and subsequent invasion of host surfaces.

Candida-induced denture stomatitis is most commonly treated with the polyene antifungal agents nystatin and amphotericin B, which are used topically and sometimes applied to the fitting surface of the denture before its insertion. Imidazoles such as ketoconazole and the newer triazoles are used for treating the condition in recalcitrant cases. With the increasing number of patients compromised by diseases such as HIV infection, haematological malignancy, and by treatment protocols, including aggressive cytotoxic and broad-spectrum antibiotic therapy, the use of other agents such as the DNA analogue 5-fluorocytosine has also become important in the management of candidosis.

Despite the availability of a range of antifungal agents for the treatment of oropharyngeal candidosis, failure of therapy is not uncommon, as the efficacy of treatment is dependent upon many factors. In the mouth the diluent effect of saliva and the cleansing action of the oral musculature often tend to reduce the viability of the agents to below that of the effective therapeutic concentration. Thus the organisms experience only a limited exposure to the antifungal agent during treatment and the concentration of the drug may vary in different niches of the mouth. Candida biofilms on oral surfaces and prosthetic devices may also contribute to failure of drug therapy.

Though there have been studies on candidal adhesion to denture acrylic pretreated with antifungal agents, there is limited information on the adhesion of the drug-exposed yeasts to denture acrylic. Thus, our main aim now was to compare the adhesion of seven oral C. albicans isolates to denture acrylic after limited exposure (1 h) of the isolates to subcidal concentrations of five antifungal agents, nystatin, amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole.

Seven isolates of oral C. albicans were studied. BM 20617, BS 742, BU 1010, BU 47204 and CA 0202 were all oral isolates from HIV-infected patients attending the Oral HIV Research Clinic at the Faculty of Dentistry, Prince Philip Dental Hospital, University of Hong Kong. These patients were not on any antimycotic therapy. The isolates designated 128981 LA and 106083 were from HIV-negative patients. The organisms were identified by the germ-tube test and the commercially available API 20 (API System, Vercieu, France) identification kits. Stock cultures were maintained at ?20°C. After recovery these were maintained on Sabouraud dextrose agar, stored at 4–6°C, during the experimental period.

Nystatin (Sigma, USA) and amphotericin B (Sigma) were dissolved in dimethylsulphoxide and absolute ethanol (3:2 ratio). 5-Fluorocytosine (Sigma) was dissolved in sterile distilled water. Ketoconazole (Janssen, Beerse, Belgium) was dissolved in dimethylsulphoxide, and fluconazole (Pfizer, Groton, CT, USA) in absolute methanol. All agents were prepared initially as 10,000 ?g/ml solutions and stored at ?20°C before use. The antifungals were suspended/diluted in the following medium during the exposure (1 h) of yeasts: RPMI 1640 medium buffered with 0.165 M MOPS containing -glutamine and lacking sodium bicarbonate (Sigma) was dissolved in 1 litre of sterile distilled water, adjusted to pH 7.2 and filter-sterilized. This liquid RPMI 1640 was stored at 2–8°C for 2–3 months.

As the antifungal agents used were dissolved in dimethylsulphoxide and absolute methanol, equivalent amounts of these chemicals were tested initially to ascertain whether they had an effect on the isolates tested. The minute volumes of the chemicals used did not have any effect on yeast survival/growth when compared with the controls.

The MIC for all seven strains for all the five drugs were determined by the broth-dilution technique, by performing 2-fold serial dilutions of the drug in microtitre plates using an inoculum of 1–5×10⁵ c.f.u./ml (a cell suspension was made at 520 nm to obtain an OD of 0.450 in sterile PBS and a 10-fold dilution was made to obtain an inoculum of 1–5×10⁵ c.f.u./ml). The MIC was determined visually and spectrophotometrically at 595 nm, after 24 h of incubation at 37°C. The MIC was defined as the lowest concentration of the drug which inhibited growth of yeast cells, as indicated by the absence of turbidity (optically clear). In determining the MIC for ketoconazole and fluconazole a slight modification of the above technique was used as these drugs exhibited a phenomenon known as trailing. Trailing occurs when the turbidity continually decreases as the concentration of the drug increases but the suspension fails to become optically clear. Thus, for ketoconazole and fluconazole the MIC was considered as the lowest concentration of the drug with a slight haziness in the well. Subcultures were made from the turbid/clear wells to confirm the turbidity results. All experiments were repeated on two separate occasions with duplicate determinations on each occasion.

The acrylic strips for the adhesion assay were prepared as described by Samaranayake and MacFarlane (1980), with some modifications. In brief, transparent self-polymerizing acrylic powder (1.5 g polymethyl methacrylate powder) was spread on an aluminium foil-covered glass slide (2.5×7.5 cm). Monomer liquid (1 ml) was poured on to the surface of the slide and immediately a second slide similar to the first was placed on top of the polymerizing mixture, and the slides were firmly secured at both ends with two binder clips. After bench-curing for 30 min the glass slides were separated. The resultant acrylic strips were cut into 5×5 mm squares, immersed in distilled water for 1 week to leach excess monomer, disinfected by dipping in 70% alcohol for 1 min, and washed with sterile distilled water. The strips were then ultrasonicated for 20 min (Branson 2200 E3 ultrasonic cleaner, CT, USA), washed again in sterile distilled water, dried and used for adhesion assay after checking their sterility.

Yeast cells, maintained on Sabouraud dextrose agar, were inoculated on to fresh plates and incubated overnight for 24 h before use. The organisms were harvested and a cell suspension prepared in sterile PBS at 520 nm to an OD of 1.5. From this cell suspension, 0.5 ml was added to tubes containing 2 ml of RPMI broth (control) and 2 ml of RPMI/drug solution (test) in which the drug concentrations varied from four–eight times the MIC. The drug concentrations used were subcidal concentrations of nystatin (×6 MIC), amphotericin B (×8 MIC), 5-fluorocytosine (×8 MIC), ketoconazole (×4 MIC) and fluconazole (×4 MIC). This gave a suspension of 10⁶–10⁷ cells/ml in each assay tube.

The tubes were then incubated at 37°C for 1 h in a rotary incubator. Following this limited exposure the drugs were removed by two cycles of centrifugation for 10 min at 3000×g. Afterwards the supernatant was completely decanted and the pellets were resuspended in 2.5 ml of sterile PBS. Viable counts of the control and the test were performed by spiral plating after drug removal, and control suspensions were reconstituted as needed to obtain a cell concentration similar to the test.