CLINICAL RESEARCH

The Effect of a Denture Adhesive on the Colonization of Candida Species In Vivo

Eunghwan Kim, DDS, MS,1 Carl F. Driscoll, DMD,2 and Glenn E. Minah, DDS, PhD3
Purpose: The purpose of this study was to evaluate the effect of a single denture adhesive on oral quantities of Candida species in vivo by determination of absolute and proportional counts of Candida species on dentures and in saliva of individuals who used this denture adhesive for a period of 14 days. Materials and Methods: Samples were collected from saliva and maxillary dentures of 12 patients who wore existing dentures without adhesives for 2 weeks, then wore dentures with adhesive (Poly Grip Free; Glaxo Smith Kline, U.K.) for 2 weeks. Periodically, maxillary dentures were sampled by adding saline to the intaglio surface, dispersing by sonication and removing aliquots for culturing. These aliquots and saliva were diluted and plated in duplicate on bismuth sulte, glycerine, and glucose yeast agar (Biggy) for recovery of Candida and on trypticase soy agar for total viable counts (TVC) of the microbiota. After 72 hours of incubation at 37C in air with 10% CO2, colony-forming units were enumerated. In each individual, absolute counts of Candida and TVC, and proportional counts of Candida relative to TVC were compared and statistically evaluated during the periods of no adhesive use (control) and adhesive use (test). Results: There were no statistically signicant differences between the test and control periods for recovery of total Candida or TVC in saliva or on dentures, or the percent of Candida relative to TVC in saliva or on dentures. Conclusion: Within the limitations of this study, the data suggested that the denture adhesive tested did not signicantly alter the denture microbiota during the 14-day trial period. J Prosthodont 2003;12:187-191. Copyright 2003 by The American College of Prosthodontists. INDEX WORDS: denture, denture adhesive, saliva, denture stomatitis, ill-tting denture

F THE 20% of the adult population in the United States who wear removable prostheses, at least 22% use denture adhesives.1,2 Between 1996 and 1997, 55 million units of denture adhesives were sold in the United States.3 Potential benets of denture adhesives are reported to be (1) improved chewing ability, (2) increased denture stability, (3) improved comfort and condence, (4)
From the Baltimore College of Dental Surgery, University of Maryland, Baltimore, MD. 1 Former Resident, Advanced Education Program in Prosthodontics. 2 Associate Professor, Director, Advanced Education Program in Prosthodontics. 3 Professor, Department of Oral and Craniofacial Biological Science. Accepted April 2, 2003. Correspondence to: Dr. Carl F. Driscoll, Room 3 D 08, Restorative Department, Baltimore College of Dental Surgery, University of Maryland, 666 West Baltimore Street, Baltimore, MD 21021. E-mail: CFD001@dental.umaryland.edu. Copyright 2003 by The American College of Prosthodontists 1059-941X/03/1203-0000$30.00/0 doi:10.1016/S1059-941X(03)00050-0

reduced food collection under dentures, (5) alleviation of denture sore spots, (6) improved mastication, (7) use in denture postinsertion care, and (8) use in patients with various medical or oral conditions including denture stomatitis.4-6 The question of what effect denture adhesive may have on the oral microora is especially important in those patients with denture stomatitis, which may involve a microbial component. Denture stomatitis has been associated with Candida albicans, which colonize and commonly reside on acrylic surfaces of dentures, especially on the intaglio surface of the maxillary denture.7,8 Denture stomatitis is also associated with denture trauma, which may be alleviated to some extent by using a soft lining material or denture adhesive.6 Relining of dentures with soft liners is generally preferred over using a denture adhesive for retention and stability, especially with an immediate or transitional prosthesis.9 Unsealed soft liners, however, showed increased colonization of Candida compared with those sealed 187

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with an acrylic varnish, implying that porosity of soft liners may result in the amplied yeast loads.10 The soft lining material has been reported to increase Candida species populations, owing in part to its porosity,11,12 but its potential to support or inhibit microbial growth is not fully understood. This question has been examined in vitro by Bartels,14 Stafford and Russell,15 and Makihira et al,16 and in vivo by Scher et al.17 Bartels14 and Stafford and Russell15 found no inhibitory effects on oral bacteria, represented as saliva samples or pure cultures of Candida species. Stafford and Russell15 did, however, demonstrate that a medium made of denture adhesive powder and 1% sucrose could support growth of Streptococcus mitis and Candida albicans, but not Neisseria pharynges. Makihira et al,16 on the other hand, found 2 of 6 brands of denture adhesive to be inhibitory to Candida albicans and 1 brand to be inhibitory to Candida tropicalis, which they attributed in part to the low pH of the denture adhesives. In an in vivo investigation, Scher et al17 reported that denture stomatitis was decreased with denture adhesives with reduction of Candida albicans, regardless of whether or not amphotericin had been incorporated into the denture adhesive. It was concluded that the denture adhesives had no positive effects on the growth of Candida species and that reduction of Candida species was probably related to improvement of the inammatory conditions. The purpose of the present study was to determine the in vivo effect on the denture microbiota of a commonly used denture adhesive in denture patients during a 14-day period of customary use by comparing absolute and proportional counts of Candida species on dentures and in saliva of individuals who used this denture adhesive. Our primary interest was the denture population at the University of Maryland Dental School that was supplied with the Poly Grip Free (Glaxo Smith Kline, U.K.) adhesive, and thus we focused our efforts on that single brand. A broad survey of other popular brands may be of interest despite the difculty in obtaining formulas from manufacturers. Ellis et al18 analyzed the constituents of several adhesives by infrared spectroscopy, and found that all contained mild antimicrobials, ingredients that swell in the presence of moisture, and additives that affect such properties as avoring or surface tension.

Materials and Methods

A group of 12 healthy subjects (4 males and 8 females: average age, 68.6; range, 65 to 76) were selected from a population of patients who had been wearing maxillary complete dentures, independent of the type of the opposing arch, for at least 6 months without denture adhesive. All of the subjects were in good general health, had no signicant medical history, and had not received antibiotics for the previous 6 weeks. Age and gender were not selection criteria. All subjects received intraoral and extraoral examinations. There were no abnormal ndings of the tongue, palate, cheek, oor of the mouth, or nasopharyngeal area. All subjects were screened for the presence of Candida species in saliva. Only subjects positive for Candida species were included in this study. Of 13 screened subjects, only 1 subject was negative for Candida species. Saliva was sampled because it is easy to collect and represents a general indicator of oral colonization by Candida. Dentures of selected participants were evaluated for stability and retention19 and were found to be clinically adequate, with no signs of palatal inammation. Most of the dentures were fabricated by the University of Maryland Postgraduate Prosthodontic Program and had not been relined before the study began. Before selection, each subject signed a consent form approved by the Institutional Review Board of the University of Maryland at Baltimore, which included a questionnaire to determine his or her comprehension of the experimental procedures. A 2-mL specimen of saliva was collected by having the subject expectorate into a sterile cup. Saliva was cultivated on days 0, 7, 14, 21, and 28 for the presence of Candida species and total bacteria. Cultivation of the same microbial categories on the patients dentures was conducted on days 0, 14, and 28. Sampling was conducted at either 10:00 A.M. or 2:00 P.M., and attempts were made to reschedule individuals at the same time for subsequent sample collection. Specimens were placed in enclosed plastic containers for transport to the laboratory and were processed for cultivation within 1 hour of collection. Subjects were encouraged to clean dentures daily and received instruction on using a denture brush (John O. Butler, Chicago, IL). Only water, with no denture cleansers or soap, was allowed during the testing period. For the rst 2 weeks, subjects did not use any denture adhesive. Starting on day 14, each subject applied the denture adhesive (Poly Grip Free denture adhesive cream) to the maxillary denture twice a day for an additional 2 weeks. On days 0, 14 (after sampling), and 28 (after sampling), each denture was cleansed using the soft denture brush with water and then thoroughly rinsed with water. Microbial sampling of dentures rst entailed dispersal of the denture biolm by sonication for 2 minutes (Kontes Cell-Disruptor, Kontes, Hazelwood, NJ) with a solution of 5.0 mL of 0.85% sterile sodium chloride solution

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(normal saline) that was placed in the intaglio of the maxillary denture. A 0.1-mL aliquot was then removed by micropipette and serially diluted to 105 by 10-fold dilution in normal sterilized saline. Cultivation procedures were similar to those used by Olan-Rodriquez et al.10 Appropriate dilutions were plated in duplicate on trypticase soy agar (TSA; Difco, Detroit, MI), a nonselective medium for determination of total viable counts (TVC), and on bismuth sulte, glycine, glucose yeast extract agar (Biggy; Difco, Detroit, MI) for recovery of Candida species. Several species of Candida could be tentatively identied on the latter medium by colony morphology. After 72 hours of incubation in air containing 10.0% CO2 at 37C, total colony-forming (TCF) units on all countable agar plates were enumerated using a stereomicroscope (American Optical, Buffalo, NY) and colony-counting device (American Optical). Saliva was diluted, plated, and cultured using the same dilution procedures, media, and cultivation technique as for the denture sample. Initial screening for Candida species was conducted by triple-streaking a sterile wire loop sample of saliva on Biggy medium, which was then incubated as previously described. Microbiological data from a sample of each subject was recorded in tabulated formats as absolute counts of Candida species per mL of saline diluent on Biggy plates and TVCs of recoverable oral bacteria per mL of diluent on TSA plates. The percentage of Candida species relative to TVC was also recorded. Data were entered on an Excel computer le, transformed into logarithms of base 10, and analyzed statistically by SPSS by Sigma Stat 2.0 (Jandel, San Rafael, CA). To test for the differences in Candida species and total viable counts from dentures or saliva during periods, with (test) or without (control) denture adhesives, a matchedpairs t test was used. The same test was used in comparisons of counts from dentures versus saliva. The effect of denture adhesives on the percentage of Candida species was analyzed by the Wilcoxon signed rank test. With a p value of 0.05 considered signicant.

Table 2. Statistical analysis of TVC N Saliva/mL Without adhesive With adhesive Denture/mL Without adhesive With adhesive
*Matched-pair t test.

Mean log10 7.46 7.47 6.98 7.27

SD log10 0.40 0.29 0.64 0.49

Signicance (p value)* 0.852 0.16

12 12 12 12

Results
Mean absolute counts of Candida species in either saliva or dentures during test or control periods are presented in Table 1. In saliva, mean values were similar during test and control periods, and differences were nonsignicant. Denture values for total Candida species were higher during the test period, but test versus control comparisons were nonsignificant. Candida species were found in signicantly greater numbers on dentures than in saliva. In both denture samples and saliva, mean TVC values were slightly higher during the no-adhesive period, but statistical differences were nonsignicant for both datasets (Table 2). Salivary TVCs were signicantly higher than denture TVCs. Mean values for the proportion of Candida species relative to TVC were higher in dentures during the control period compared to the test period, but the differences were not statistically signicant (Table 3). Test versus control salivary proportions were nonsignicant. Values for absolute counts of Candida species per mL of saliva were considerably lower than counts per mL of denture uid, and the differences were signicant (p 0.007) (Table 4).

Table 1. Statistical Analysis of Absolute Counts of Candida Species N Saliva/mL Without adhesive With adhesive Denture/mL Without adhesive With adhesive 12 12 12 12 Mean log10 3.4 3.4 4.3 4.2 SD log10 2.0 1.8 1.5 2.1 Signicance (p value) 0.93* 0.97

Table 3. Statistical Analysis of the Percentage of Candida Species Relative to TVC N Saliva/mL Without adhesive With adhesive Denture/mL Without adhesive With adhesive 12 12 12 Mean SD Signicance (p value)* 0.625 0.37

0.18 35 0.18 0.29 2.33 2.21 4.57 4.23

*Matched-pair t test. Wilcoxon signed-rank test.

*Wilcoxon signed-rank test

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Table 4. Comparison of Absolute Counts of Candida Species in Saliva and on Dentures, With and Without Denture Adhesive Saliva Mean log10 Without adhesive With adhesive
*Matched-pair t test.

Saliva SD log10 2.0 1.8

Denture Mean log10 4.26 4.20

Denture SD log10 1.5 2.0

Saliva Versus Denture Signicance (p value)* 0.007 0.007

3.4 3.4

Discussion
This present study has demonstrated that using a denture adhesive for 14 days produced no statistically signicant increase or decrease of Candida or absolute counts of microorganisms in the oral cavity or on denture intaglio surfaces, compared with a similar period without using adhesive. Although the data showed increases or decreases of microorganisms with or without denture adhesives in individual subjects, no general trends were seen. The percentage of Candida relative to TVC on dentures showed similar statistical results. All colonies on countable Biggy plates appeared consistent with the morphology of Candida albicans, but were not conrmed by more in-depth identication. Although the present research suggests that denture adhesives may not directly affect oral quantities of Candida species or increase total bacteria, researchers have previously assumed that proliferation of microorganisms would be enhanced due to the stickiness of adhesives, or their stagnation if not replaced often. However, regularly removing the adhesive and washing the intaglio surfaces of their dentures before applying a new lm may serve to reduce the microbial buildup. In the present study, the numbers of Candida and TVC of most subjects showed decreases during the rst 2 weeks of the trial (no adhesives). This nding might indicate that subjects were motivated to follow diligent denture care procedures as a result of participation in the study. Because most of the subjects dentures had been fabricated within 1-1/2 years, the dentures had proper retention and stability, with adequate t between the intaglio surfaces of dentures and the tissue surface. This would suggest that the denture adhesive lm was thin. Most patients who use denture adhesives, however, do so because of retention and stability problems, implying thicker denture adhesive lms than in the present study. This, in addition to inammatory conditions, may produce different effects on the microbiota. The previous in

vivo study of denture adhesives in patients with denture stomatitis by Sher et al,17 however, found that adhesives were associated with reduced Candida levels, due presumably to reduced trauma and thus decreased inammation of the mucosal tissue. Clinical implications of Candida colonization of denture adhesives may be evident in patients who seek relief from denture stomatitis or who wear transitional prosthesis. If adhesives stimulate Candida proliferation, then they could exacerbate tissue inammation in the case of denture stomatitis.7,8 Although not directly addressed in the present investigation, it appears that regularly replacing denture adhesive after removing the old adhesive may aid control of the denture biolm. For this reason, it may be also be advantageous from a microbiological standpoint with immediate or transitional prostheses. Limitations of the present investigation were the absence of multiple brands of denture adhesives, small study population, and relatively short trial period. Future studies should include longitudinal assessment of yeast proliferation over a longer period. Long-term use of ill-tting dentures should also be considered, to evaluate the effects of prothesis quality and thereby the lm thickness of adhesives. Makihira et al16 observed differences in pH of Candida cultures grown on commercial denture adhesives, all of which exhibited a pH 6.0 before exposure to Candida. It was observed that a pH between 5 and neutral may stimulate Candida proliferation, and also may be a risk factor for cementum demineralization. Several denture adhesives had a sufciently low pH (3) to potentially inhibit Candida. The pH of Poly Grip Free was found to be 5.92 0.36 after hydration but before oral exposure. In summary, the ndings of our study indicate that denture adhesive can be used safely for a short period, if patients are diligent in denture-cleansing procedures.

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Conclusions
Within the connes of this study, the following conclusions can be drawn: 1. The denture adhesive cream (Poly Grip Free) did not affect the colonization of Candida species as measured by absolute and proportional counts, or absolute counts of bacteria on the surface of the dentures or in the saliva during a short-term (14-day) study period. 2. Patient hygiene may have been a factor in the lack of signicant microbial changes. Daily cleaning of dentures by the subjects or daily replacement of the adhesive may have contributed to the lack of signicant microbial changes noted.

References
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6. Adisman K: The use of dental adhesives as an aid to denture treatment. J Prosthet Dent 1989;62:711-715 7. Webb BC, Thomas CJ, Willcox MD, et al: Candida-associated denture stomatitis. Aetiology and management: A review. Part 1. Factors inuencing distribution of Candida species in the oral cavity. Aust Dent J 1998;43:45-50 8. Radford DR, Challacombe SJ, Walter JD: Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro. Crit Rev Oral Biol Med 1999;10:99-116 9. Slaughter A, Katz RV, Grasso JE: Professional attitudes toward denture adhesives: A Delphi technique survey of academic prosthodontists. J Prosthet Dent 1999;82:80-99 10. Olan-Rodriguez L, Minah GE, Driscoll CF: Candida albicans colonization of surface-sealed interim soft liners. J Prosthodont 2000;9:184-188 11. Imai Y, Tamaki Y: Measurement of adsorption of salivary proteins onto soft denture lining materials. J Prosthet Dent 1999;82:348-351 12. Samaranayake LP, MacFarlane TW: An in vitro study of the adherence of Candida albicans to acrylic surfaces. Arch Oral Biol 1980;25:603-609 13. Waters MG, Jagger RG: Water absorption of RTV silicone denture soft lining material. J Dent 1996;24:105-108 14. Bartels H: Bacteriological appraisal of adhesive denture powders. J Dent Res 1945;24:15-16 15. Stafford GD, Russell C: Efciency of denture adhesives and their possible inuence on oral microorganisms. J Dent Res 1971;50:832-836 16. Makihira S, Nikawa H, Satonobu SV, et al: Growth of Candida species on commercial denture adhesive in vitro. Int J Prosthodont 2001;14:48-52 17. Scher EA, Ritchie GM, Flowers DJ: Antimycotic denture adhesive in treatment of denture stomatitis. J Prosthet Dent 1978;40:622-627 18. Ellis B, El-Nakash S, Lamb DJ: The composition and rheology of denture adhesives. J Dent 1980;8:109-118 19. Olshan AM, Ross NM, Mankodi S, et al: A modied Kapur scale for evaluating denture retention and stability: Methodology study. Am J Dent 1992;5:88-90