Journal of Dentistry, 13, No. 4, 1985,
Printed in Great Britain
The effect of miconazole on palatal candidosis induced in the Wistar rat M. M. Norris,* BDS, FDS D. J. Lamb,t BDS, FDS, MDS G. T. Craig,* BDS, PhD, FDS, MRCPath *Department
of Oral Pathology
and t Restorative
M. V. Martin, BDS, BA PhD Department
ABSTRACT Autopolymerizing acrylic resin supplemented with 10 per cent miconazole nitrate was tested as a treatment for palatal candidosis as induced by Shakir et aL (198 1) in the Wistar rat. It was found that by fitting rats with appliances supplemented with 10 per cent( w/w) miconazole in the polymer powder, palatal candidosis could be prevented, and previously infected animals were cured after therapy for one month Rata fitted with supplemented appliances showed no adverse side-effects and tended to gain weight more rapidly than animals with unsupplemented appliances.
INTRODUCTION Dentureinduced stomatitis is a common superficial fungal infection of the denture-bearing tissues. Can&da albicuns is the organism thought to be responsible for the disease (Cawson, 1966; Budtz-Jorgensen, 1974). The prognosis for conventional treatment is often unsatisfactory, and relapse is frequent (Holbrook and Kippax, 1979; Bergendal, 1982). Saliva quickly washes away topically applied drugs and limits the period of effective drug therapy. Consequently, in order to provide a period of sustained local therapy, antifungal agents have been incorporated into denture-lining materials, though generally with adverse effects on their physical properties (Douglas and Clarke, 1975; Thomas and Nutt, 1978). Miconazole (Janssen Pharmaceuticals Ltd, Marlow, Bucks.) is an antifungal agent that appears to be effective and well tolerated when applied topically (Brincker, 1976; Roe& Petersen, 1978). Recent studies have shown it to be released readily from autopolymerizing acrylic when immersed in water (Norris et al., 1984). The aim of our study was to examine the therapeutic potential of miconazolesupplemented autopolymerizing acrylic resin for the treatment of palatal candidosis. The rat model of Shakir et al. (1981) was used in the investigation, and miconazole tested in both a preventive and curative mode.
Appliances Palatal appliances similar to those described by Shakir et al. (1981) were made from autopolymerizing acrylic resin (Simplex Rapid, Howmedica, London). The monomer and
Norris et al.: Miconazole effect on palatal candidosis
polymer were mixed in the ratio 3 : 5 (v/w) and cured in a hydroflask (Kulzer and Co., FR Germany) for 10 min. Miconazolesupplemented appliances were made in the same way, but first 10 per cent (w/w) miconazole nitrate was added to the polymer powder and mixed thoroughly before adding it to the monomer liquid. Inoculation Candida a&cans 3091 (obtained from the National Mycological Reference Laboratory, London) was grown on Sabouraud’s agar for 24 h at 37 “C, then harvested by scraping from the surface, taking care to avoid removing any agar. Immediately before insertion, the fitting surface of the appliance was smeared with approximately 30 mg (wet weight) of the harvested C. albicans, a dose equivalent to about 5 X lo8 blastospores. Test
To test the preventive effect, 26 male Wistar rats (mean weight 324 g) were anaesthetized and inoculated orally with C. albicans 309 1 on an appliance cemented to the upper incisors with autopolymerizing acrylic resin. Fourteen animals were fitted with miconazolesupplemented appliances and the other 12 were fitted with unsupplemented appliances. The animals were given food and water ad fibifum weighed twice weekly and their mean weekly weights calculated Four weeks later, all the animals were killed by intraperitoneal injection of a lethal dose (> 120 mg/kg body weight) of Veterinary Nembutal (Abbot Laboratories Ltd, Kent). The appliances were removed and the rats’ palates swabbed with sterile swabs (Exogen Ltd, Clydebank) before the palatal tissues were removed for histological examination. To test the curative effect, 37 male Wistar rats (mean weight 315 g) were anaesthetized, fitted with an inoculated unsupplemented appliance and given food and water as before. Four weeks later when palatal candidosis should have been induced, five of the animals were killed as controls and their palates swabbed before removal for histological examination. The remaining animals were now anaesthetized, their palates swabbed and new inoculated appliances fitted. Sixteen animals were given miconazolesupplemented appliances and the remaining 14 fitted with unsupplemented appliances. A further four weeks later (i. e., eight weeks after the start of the experiment), all the animals were killed. Their palates were swabbed then removed for histological examination. Microbiology Swabs of the rats’ palates were plated out on to Sabouraud’s agar and incubated for 72 h at 37 “C. Yeast colonies were identified as being C. albicans on the evidence of their Gramstained appearance, germ-tube production and chlamydospore formation( Winner and Hurley, 1966). Histology To ensure that the tissue examined from each animal was representative, a systematic random sampling strategy was adopted for the palate of each animal. At regular intervals throughout each specimen pairs of serial sections were taken. In total, 50 pairs of sections were taken from
Journal of Dentistry,
Fig. 7. Rat appliances; unsupplemented (u). Table /. Reasons for exclusion
No. started Reason for exclusion Appliances lost Excess weight loss Anaesthetic death No. completed
and curative exDeriments
Curative expt Unsupple mented
4 0 1 12
; 0 11
5 :, 8
animals from the preventive
Preventive expt SuppleUnsupplernented mented
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each palate, and one section of each pair was stained with haematoxylin and eosin, the other with periodic acidschiff. The criteria taken as indicative of a diagnosis of candidosis were: 1. Evidence of fungal invasion of the superficial keratin and epithelial layers. 2. Epithelial hyperplasia. 3. Intra-epithelial neutrophil micro-abscess formation and subepithelial inflammation.
RESULTS AND DISCUSSION For a variety of reasons, approximately one-third of the animals beginning the experiment were excluded during its course ( Table I). Nineteen completed the preventive experiment and 27 the curative experiment. The most common reason for exclusion was fracture of the appliance, fracture often following deep grooving of the appliance by abrasion against the lower incisor teeth.
Norris et al.: Miconazole effect on palatal candidosis
Fig. 2. Preventive experiment: Rat fitted with supplemented appliance. The epithelium and connective tissue appear relatively normal. H 8 E, x 32.
Fig. 3. Preventive experiments Rat fitted with unsupplemented appliance. a, Disrupted keratin covers hyperplastic and oedematous epithelium which is infiltrated throughout all layers by inflammatory cells; the underlying connective tissue is notably vascular and heavily inflamed. H Et E. x 32. b Adjacent sectron to that shown in a stained to reveal fungal hyphae wrthin the keratin layer and penetrating the epithelium. The appearances in a and b are consistent with those of candidosis. PAS, x 64.
More supplemented appliances (9) were lost in this way than unsupplemented (4). This was not thought to be due entirely to differences in physical properties (compression tests on cylinders of supplemented and unsupplemented resin showed no significant differences in modulus of elasticity) but in part due to the opacity of miconazolesupplemented acrylic (Fig. I). For this reason any small subsurface defects in the appliances could not be detected and the risk of fitting defective appliances had to be taken. The results of the experiment to test prevention are given in Table II and show that none of the animals wearing miconazolesupplemented appliances showed microbiological or histological evidence of candidosis. While the palatal epithelium of rats wearing supplemented appliances (Fig. 2) closely resembled that of normal rats, a majority of those wearing unsupplemented appliances showed clear evidence of candidosis (Figs 3 a, b). Fischer’s exact test showed the difference to be significant (P < O-01 7). Both supplemented and unsupple
Journal of Dentistry, Vol. 1 ~/NO. 4 (1985)
Supplemented (n Unsupplemented
( n = 11 I
Fig. 5. Curative experiment: rat fitted with SUP
Week of experiment Fig 4. Preventive experiment: bar chart showing mean weekly weight of animals
plemented appliance. The epithelium appears only slightly thickened but shows marked hlyperkeratosis and accumulation of debris superfic:ially. t-l Et E, x 32.
Table //. Results of experiment to test prevention Supplemented* No. completed Swabs +ve Histology +ve
Unsupplementeff 11 3 6
*A significant difference exists between plemented groups, P < 0.017.
mented groups of rats remained in good condition and started to regain weight soon after the anaesthetic. Rats wearing supplemented appliances gained weight more rapidly than those wearing unsupplemented appliances, but the difference was not significant (Fig. 4). The results of the curative experiment are shown in Table III. Again, using the same statistical test as before, a significant difference was detected between the supplemented and unsupplemented groups (P < O-003). No rats wearing supplemented appliances showed histological evidence of candidosis, although much surface debris (mainly squames) was present and the epithelium was hyperkeratotic in places (Fig. 5). About half the animals wearing unsupplemented appliances showed evidence of candidosis (Fig. 6). Only one rat in the supplemented group showed microbiological evidence of the presence of C. albicans, and although rats wearing supplemented appliances gained slightly more weight than those wearing unsupplemented appliances, the difference was not significant (Fig. 7).
Norris et al.: Miconazole
effect on palatal candidosis
Supplemented( n: 121 Unsupplemented( n
Fig. 6. Curative experiment: rat fitted with unsup plemented appliance. lntraepithelial neutrophil microabscess formation is evident beneath the keratin layer; the remaining features resemble those shown in fig. 3a and the appearance overall is that of candidosis. H Et E, x 64.
Curative experiment: bar chart mean weekly weight of animals.
Tab/e 111.Results of experiment to test cure For the first four weeks all animals wore unsupplemented appliances in order to induce palatal candidosis Supplementedl No. completed Swabs +ve (4 weeks) Histology +ve (4 weeks) Swabs +ve (8 weeks) Histology +ve (8 weeks)
*Significant difference exists mented groups, P < 0.003.
Lamb and Martin ( 1983) have investigated recently the treatment of palatal candidosis in the rat by use of an appliance supplemented with chlorhexidine acetate. While being treated effectively, rats wearing supplemented appliances lost weight, this effect is ascribed to the taste of chlorhexidine being as unpleasant to rats as it is to human beings. Miconazole presents none stomatitis, of these problems when testing in viva and if used to treat human dentureinduced in the form of a drug-supplemented m-line, may be the therapeutic agent of choice.
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Vol. 1 ~/NO. 4 (1985)
CONCLUSIONS 1. The use of an autopolymerizing acrylic resin appliance supplemented with miconazole is an effective way of preventing or curing palatal candidosis in the rat. 2. Miconazole is well tolerated, and miconazolesupplemented autopolymerizing acrylic resin in the form of a denture reline may well be the method of choice for the treatment of denture-induced stomatitis.
Acknowledgements We wish to thank Professor C. J. Smith of the Department of Oral Pathology, University of Sheffield, for his aid with the histopathological assessment of the specimens, and Dr G. Constable, Department of Probability and Statistics, University of Sheffield, for statistical aid We are also grateful to Trent Regional Health Authority for their financial support and Janssen Pharmaceuticals for the provision of materials.
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Norris M. M., Martin M. V. and Lamb D. J. (1984) Release into water of miconazole and ketoconazole from autopolymerizing acrylic resin discs. J. Dent. Rex 63, 501. Roed-Petersen B. (1978) Miconazole in the treatment of oral candidiasis. Int. J. Oral Surg. 7, 558. Shakir B. S., Martin M. V. and Smith C. J. ( 1981) Induced palatal candidosis in the Wistar rat Arch.. Oral Biol. 26, 187.
Thomas C. J. and Nutt G. (1978) The in vitro fungicidal properties ofViscoge1 alone and combined with nystatin and amphotericin B. J. Oral RehabiI. 5, 167. Winner H. I. and Hurley R (1966) Symposium on Candida Infections. Edinburgh, Livingstone.