Effectiveness of Antifungal Agents in Tissue Conditioners in Treating Candidiasis Over Time

INTRODUCTION

The prevalence of Chronic Atrophic Candidiasis has been studied extensively, since it is a common and treatable condition especially in the elderly. The prevalence ranges from 11-67% in complete denture wearing populations.² Candidiasis is a major problem especially for institutionalized denture wearers, many of whom have reduced manual dexterity, cognitive control, memory loss and reduced compliance. Chronic atrophic candidiasis can be classified as Newton Type I-localized erythematous, Type II-generalized erythematous, and Type III-hyperplastic granular, with severity of inflammation greatest in Type III patients.³ It maybe associated with burning mouth syndrome, xerostomia and immunocompromised diseases. Candidal infection and ill fitting dentures are the primary causes of oral Candidiasis.⁴

Currently, two types of treatment methods are employed to ameliorate the condition, use of topical antifungal agents or use of tissue conditioners within an existing prosthesis. In a recent in vitro study¹, antifungal agents were mixed with tissue conditioners to explore the efficacy of this method of drug delivery. The major advantage of this type of therapy is that it does not rely on patient compliance. Different combinations of tissue conditioners and antifungal agents were mixed at different weight concentrations and fashioned into cores placed in the centre of agar dishes containing cultured Candida albicans in the in vitro study¹. The recorded inhibition diameters demonstrated that this method of treatment is efficacious. The most effective combinations were 5%wt/wt itraconazole in Coe Soft and in FITT. Peak fungicidal activity was achieved after 3 days. To what extent the fungicidal effect continues beyond this point, remained unclear. It was important to clarify this point before progressing to the clinical trial stage, as it would help in developing the research methodology. Thus, the present study is a continuation of the previous in vitro study¹, with the objective to investigate the effectiveness of the antifungal and tissue conditioner combinations over time.

METHODS

Candida albicans
Clinical isolates of the yeast C. albicans were used as the test organism for antifungal activity.

C. albicans ATCC 10231, obtained from Mt. Sinai Hospital, Toronto, was cultured as described by Thomas and Nutt.⁵ In brief, yeast was grown on a stock plate at 37°C and incubated for 3 days. 2mm3 of the stock culture was then transferred and diluted with 2ml saline. 0.2ml of the diluted solution was mixed with 1.8ml of saline. This step was repeated to obtain a 10-3 solution. The number of cultures per ml of 10-3 solution was counted with a Petrof-Hausser counter. 1000 organisms per ml was accepted as the standard inoculum concentration.

Mixing antifungal agents with tissue conditioners

0.1ml of 10^-3 solution was dropped on each sterile Sabouraud agar plate and was spread out using a spreading rod. The plates were incubated at 37°C for 72 hours at humidity of 75%. 6mm diameter holes (cores) were punched in the centre of each 5mm deep agar plate. Nystatin, fluconazole and itraconazole were chosen as the test antifungal agents. These agents are commercially known as Mycostatin^TM (Bristol-Meyer Squibb), Diflucan^TM (Pfizer) and Sporanox^TM (Janssen Ortho), respectively. Nystatin was supplied in a yellowish oral suspension containing 100,000 Units per ml. Fluconazole was supplied in white oral suspension powder and when reconstituted it yields a 10mg per ml solution. However in this study, fluconazole powder for oral suspension was not reconstituted with water. Instead, the powder was used directly from the container. Itraconazole was supplied in a clear yellowish oral suspension which contained 10mg per ml.

The tissue conditioners tested were Coe Soft^TM (Coe Laboratories) and FITT^TM(Kerr). These tissue conditioners were mixed according to manufacturer's instructions. Antifungal agents were hand mixed into the tissue conditioners at 5 wt/wt% (i.e. 0.95g base + 0.05g antifungal agent). A disposable syringe was used to inject each antifungal agent and tissue conditioner mixture into the cores. The amount injected and time of injection were recorded for each plate.

Each antifungal and tissue conditioner combination was repeated three times. These combinations include mixtures of nystatin, fluconazole, or itraconazole at 5 wt/wt% with Coe Soft, or Fitt, with sterilized saliva.¹

The caliper used for the measurement of the inhibition diameter was standardized with a standard error of 0.1mm. Only the largest inhibition diameter reading ("raw data") was taken for each plate. Maximum inhibition diameter was calculated by subtracting the core diameter (6 mm) from the raw data.

Plate Transfers

Inhibition diameters were recorded until the inhibition diameter had reached a plateau. This was also the stage when the antifungal agents reached their peak activity. The cores were then transplanted into a fresh set of agar plates (also known as Transfer 1 plates). Petrof-Hausser counter was used to ensure that Transfer 1 plates had standardized amount of C. albicans pre-grown on it as with the first set of plates. As well, they had a 6mm hole punched in the centre as previously. After the first transfer, the inhibition diameters were recorded daily as before. Three days after the first transfer, the cores were transferred again to a new set of pre-grown C. albicans plates (Transfer 2 plates). These Transfer 2 plates were observed for another 3 days to investigate the fungicidal capability of the cores over time. Therefore, the plates were incubated for 11 days in total. Inhibition diameters were measured at hours 15, 24, 39, 48, 72, 135, 159, 183, 207, 231, and 255.

Statistical Analysis

One-way ANOVA and the Tukey Studentized range test were used to compare the inhibition diameters at the time of peak and minimal effect. Repeated-measures ANOVA was used to compare the inhibition diameter of different combinations of antifungal agents and tissue conditioners over time. Statistical tests were two-tailed and at the 5% significance level.

RESULTS

The statistical tests found significance (ANOVA p<0.05) in inhibition diameter measurements with different antifungal and tissue conditioner combinations.

Most Effective Combination
The most effective combinations was itraconazole in Coe Soft (mean inhibition diameter of 17.6mm, Table 1) followed by itraconazole and FITT (17.1mm) but the difference is not significant (ANOVA, p<0.05). They were significantly better (ANOVA, p<0.05) than the fluconazole and nystatin groups at all time points with the exception at 135hr. At this time point, itraconazole in Coe Soft was significantly better (ANOVA, p<0.05) than all groups, whilst itraconazole in FITT was not significantly different (ANOVA, p<0.05) from nystatin in Coe Soft or nystatin in FITT. Fluconazole and nystatin combinations had mean inhibition diameters that ranged from 6.1mm to 7.9mm. Nystatin and FITT had the least mean inhibition diameter and was significantly different from the other combinations (ANOVA, p<0.05) (Table 1).

Table 1: Mean Inhibition Diameter by Repeated Measures ANOVA

Peak Activity
Average peak activity of all combinations was 20.7mm recorded at 63.5 hours (i.e. 2.7 days) (Table 2). At the peak, both itraconazole groups were significantly better (ANOVA, p<0.05) than all other combinations. Fluconazole in Coe Soft had significantly higher antifungal activity (ANOVA, p<0.05) than fluconazole in FITT and nystatin in Coe Soft. Nystatin in FITT had the least fungicidal activity (Table 2).

Table 2: Time and Inhibition Diameter at Peak Effect

*Means with the same letter are not significantly different according to the Tukey test.

Least Activity
The least effect of a combination is the inhibition diameter recorded at time of lowest fungicidal activity. In studying the groups daily for 11 days, least effect was registered at approximately 196 hours (i.e. 8.2 days). The average effect at that time was 3.2mm
(Table 3). As well, there was no significant difference between FITT and Coe Soft combinations at the time of least effect.

Table 3: Time and Inhibition Diameter at Minimum Effect

*Means with the same letter are not significantly different according to the Tukey test.

Transfer #1
The itraconazole group had higher inhibition results throughout this first transfer period (Figure 1). At the end the first transfer period (183hr), the itraconazole group (10.0mm) and nystatin in FITT (8.0mm) had significantly higher fungicidal activity (ANOVA, p<0.05) than the other groups. There was a rise in inhibition diameter following the first transplant (135-183hr). (Figure 1).

Fig. 1 Inhibition diameters of combinations of the antifungal agents and tissue conditioners: at 5%wt/wt with saliva.

Mean inhibition diameter ranged from 2.3mm at 231 hours for nystatin in Coe Soft to 32.3mm at 72 hours for itraconazole in Coe Soft. Itraconazole had greater fungicidal activity than fluconazole and nystatin. Optimal inhibition was achieved at approximately 63.5 hours followed by a drop. There is a slight rise after the first transplant (135-183 hours) and a plateau after the second transplant (207-255 hours).

Transfer #2
In contrast to transfer 1, there was no slight rise in inhibition activity; rather there was an immediate plateau (Figure 1). For instance, itraconazole in FITT had inhibtion diameter of 5.3mm, 5.0mm, 5.3mm at 207hr, 231hr and 255hr, respectively. The itraconazole group had the highest inhibition results (6mm for itraconazole in Coe Soft at 255hr). Similar to the itraconazole group, fluconazole in FITT was also significantly better (ANOVA, p<0.05) than the other groups at 231 hours.

Interaction between antifungal activity and time
As shown from the time versus inhibition diameter figure (Figure 1), all combinations exhibited different fungicidal activitiy at different times (ANOVA, p<0.05). For example, fluconazole in Coe Soft showed some inhibitory effect before 72 hours, although not as high as itraconazole, but exhibited the lowest effect of all combinations after the plates were transferred.

DISCUSSION

In the previous study1, the most effective antifungal agent and tissue conditioner combination and the optimum concentration were found. All treatment group combinations had greater fungicidal activity than negative controls (i.e. tissue conditioner only) and comparable inhibition diameters to positive controls (i.e. antifungal agents alone). The peak fungicidal activity was also noted.¹ However, there was a need to explore the long term effectiveness of the mixtures, which has not previously been investigated by other studies.

The purpose of the current study was to examine the timing of peak activity and subsequent effects by means of transferring cores to fresh agar plates so that an initial protocol may be derived for a pilot study. In the preceding study¹, continued fungicidal activity could not be explored due to lack of fresh C. albicans in the area immediate to the core.

The plate transfer technique allowed the exhibition of continued fungicidal activity by means of having fresh C. albicans around the core. The kinetic flow characteristics of the antifungal are then controlled for, because any new activity can be readily measured. The rationale for adding sterilized saliva to the cores was to serve as a standard protocol for a controlled comparison of the antifungals used. 5% wt/wt was tested as it was the best concentration of antifungal agents in tissue conditioner found in the previous study1.

Comparing the combinations at this percentage enabled contrast of effectiveness of antifungal agent mixed in the base; as oppose to only comparing effectiveness of antifungals agents alone.

Oral suspensions of the antifungal agents were selected as in the previous study. The observation period totaled 11 days. The core was first transferred after a peak in antifungal activity was registered.

In concurrence with the previous in vitro study1, itraconazole combinations had significantly higher antifungal activity than fluconazole and nystatin groups. The most effective combination was itraconazole in Coe Soft with a mean time adjusted inhibition diameter of 17.6mm (Table 1).

FITT combinations reached the peak faster than combinations with Coe Soft, but the difference in time was not significant. The average peak of all combinations was reached at 63.5 hours. There was a rise in inhibition diameter throughout the period following the first transfer. Conversely, there was no obvious rise in inhibition results after the second transfer. These results reflect continual antifungal activity up to approximately two days after the first transfer followed by a plateau after the second transfer. The mean least effect of all combinations was 3.2mm and the mean time of least effect was recorded at 196 hours, followed by a plateau (Table 3, Figure 1). This mean inhibition diameter result was similar to results obtained by testing the negative control (tissue conditioners only) of 3.1mm in the previous in vitro study1. The fungicidal activity declined in the second transfer and this trend demonstrated a loss of fungicidal activity after 9 days.

CONCLUSION and RECOMMENDATION OF A PILOT STUDY

Inhibition diameters recorded in this study, confirmed that itraconazole groups had significantly higher fungicidal properties than fluconazole or nystatin. The peak effect was achieved after approximately 3 days. Both of these findings agreed with results of the previous study1. From the plateau of antifungal activity observed after the second transfer, it can be concluded that antifungal agent and tissue conditioner cores were effective up to 9 days after placement. Consequently, it is ideal to replace the antifungal agent and tissue conditioner combinations after 3 days when the peak activity has been reached. It is also advisable to remove the tissue conditioner within 9 days after which there is little fungicidal effect, if a single combination is placed and not replaced subsequently.

The in vitro results recorded in this study further support the efficacy of mixing antifungal agents into tissue conditioners as a method of drug delivery. The tissue conditioner method of drug delivery has the advantages of being cheaper than conventional therapy and can simultaneously relieve candidal infection and traumatized denture bearing tissues. In addition, it is not dependent on patient cooperation, which is most beneficial in the institutionalized setting.

The results of this study will be incorporated into a clinical protocol in a pilot study to evaluate the effectiveness of this modality of drug delivery.

ACKNOWLEDGEMENTS

We would like to thank Dr. Edward Fillery, Microbiology Dept., Faculty of Dentistry, Toronto for his advice on microbiology techniques, Mrs. Rita Bauer for photography and to all other individuals who helped with this project. This research was supported by MRC grant.

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