Journal of Oral Rehabilitation 2006 33; 36–42
Association of candidal activity with denture use and salivary
flow in symptom-free adults over 60 years1
K. IKEBE, K. MORII, K. MATSUDA, K. HATA & T. NOKUBI
Regeneration, Osaka University Graduate School of Dentistry, Yamadaoka Suita Osaka, Japan
SUMMARY
The purpose of this study was to examine the
amount of palatal mucosa Candida species asso-
ciated with denture use and stimulated salivary
flow in symptom-free adults over 60 years. The
subjects were 351 (189 men and 162 women)
independently living elderly people with a mean
age of 66Æ7
4Æ3 (SD) years. Candidal activity of
palatal mucosa was evaluated by the pH change in
the medium that was associated with the acid
production of the yeast. Subjects whose stimulated
salivary flow rate was less than 0.5ml/min were
placed in the hyposalivation group. A multiple
logistic regression analysis was used to determine
if an independent variable was statistically signifi-
cant after controlling for other variables. Candidal
activity of the palatal mucosa was significantly
associated with the dental status of the maxillae
(Kruskal-Wallis test, P < 0Æ001), but was not signifi-
cantly associated with age or drug intake. In
Introduction
The most common infection involving oral mucosal
tissues in the elderly patient is candidosis. Candida
species are typically harmless and common fungi of
the surface of the oral mucous membrane (1). Com-
mensal existence of intraoral Candida species varies
from 20 to 50% in a healthy dentulous population (2,
3) and up to 75% in a population wearing dentures
(3, 4). Although most oral candidoses are symptom-
less, they can indicate the presence of an underlying
1
This paper was presented at the 80th International Association of
Dental Research General Session in San Diego, USA in 2002.
ª 2006 Blackwell Publishing Ltd
Division of Oromaxillofacial
maxillary denture wearers, Candidal activity of
palatal mucosa had a significantly positive correla-
tion with candidal activity of tissue fitting surfaces
of maxillary dentures (r ¼ 0Æ806, P < 0Æ001). A multi-
ple logistic regression analysis showed that high
candidal activity of the palate was significantly
associated with being male and wearing maxillary
removable dentures. Stimulated salivary flow rate
was likely to be negatively related to high candidal
activity (P ¼ 0Æ07). This study suggests that the
activity of Candida species in the oral cavity is
associated with the wearing of removable dentures
and stimulated salivary flow, independent of age or
gender even in the relatively healthy elderly.
KEYWORDS: Candida, palatal mucosa, salivary flow,
removable denture, independently living elderly,
age, symptom-free, multiple logistic regression ana-
lysis, geriatric dentistry, epidemiology
Accepted for publication 4 April 2005
systemic disease (5, 6). Certain systemic conditions
(e.g. diabetes), defects in the immune system, and/or
some medications (e.g. antibiotics, corticosteroids) may
predispose the transformation of a benign coloniza-
tion, such as Candida species, into parasitic pathogens
(7). Therefore, candidosis mainly has been discussed
regarding a cohort of the frail elderly (8–11), not for
physiologically aged, comparatively healthy adults. In
Japan, most old people (95% between 75 and
79 years, 90% between 80 and 84 years, and 80% of
those 85 years and older) are functionally independent
and have no limitations in their daily activities (12).
Denture use and hyposalivation are common not only
in the frail elderly, but also in the comparatively
36
 ASSOCIATION OF CANDIDAL ACTIVITY WITH DENTURE AND SALIVA
healthy elderly who live independently. The 1999
Japanese national survey of dental diseases by the
Ministry of Health and Welfare collected data from
every member of all households in 300 municipalities
that had been randomly sampled in every prefecture
(13). The survey found that the number of residual
teeth gradually declined after age 50. Consequently,
50% of people between 65 and 74 years and 70% of
those between 75 and 84 used removable dentures.
Another change in the oral cavity brought on by
ageing is oral dryness. It is well known that saliva
plays an important role in the preservation and
maintenance of oral health and function (14). The
prevalence of dry mouth varies from 13 to 39% in
independent or community-living old people (15–20).
In the similar subjects of this study, it was demon-
strated that approximately one-fourth were classified
as having hyposalivation (21).
The purpose of this study was to examine the amount
of palatal mucosa Candida associated with denture use
and stimulated salivary flow in symptom-free adults
over 60 years.
Methods
Study population
The subjects in this study were participants of the Senior
Citizens’ College of Osaka prefecture. The study sample
consisted of community-dwelling, independently living
elderly people over the age of 60 years who attended
lectures once a week. This college is one of the adult
educational systems supported by the government of
Osaka prefecture, which enrols volunteers for a period
of 1 year. This course focused not only on health topics
but also on other topics of interest to elderly people,
such as finance or culture.
The research protocol was approved by the Institu-
tional Review Board of Osaka University Graduate
School of Dentistry. After informed consent, subjects
were given the opportunity to ask questions while
completing the questionnaires; subjects could refuse to
participate in the oral examination portion of the study.
The final study population who voluntarily partici-
pated in the dental and oral examinations consisted of
351 persons (189 men and 162 women) with a
mean age of 66Æ7
4Æ3 (s.d.) years. Information about
the use of prescribed drugs was sought during the
interview.
ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 36–42
37
Dental status
Subjects were classified into three categories according
to the dental status of their maxilla: complete denture,
removable partial denture, or natural dentition.
Candidal activity
Samples of palatal mucosa were collected with sterile
swabs because stomatitis is most prevalent on the
maxillary hard palate (22). In the subjects whose
palates were covered with removable dentures, samples
of tissue fitting surfaces of a maxillary denture were
also collected. Persons with a symptom or an obvious
sign of stomatitis in the palatal mucosa were excluded
from the analyses.
Samples were incubated for 24h in a liquid selective
medium for Candida species (StomastatTM)* (23). Cand-
idal activity was evaluated by the pH change in the
medium that was associated with the acid production of
the yeasts. Lower pH of the medium indicates higher
candidal activity.
Saliva collection
Stimulated whole saliva was collected by the mastica-
tion method (24). Subjects with complete or removable
partial dentures wore their dentures during the saliva
collection period (20). Subjects were asked to swallow
all the saliva in the mouth, then to chew the wax for
2 min at their own pace and spit into a graduated tube.
All saliva specimens were collected between 10:00 AM
and 3:00 PM.
Subjects were classified into two groups according to
stimulated salivary flow rates. Persons whose stimula-
ted flow rate was <0Æ5 mL min)1 were placed in the
hyposalivation group, and the rest were designated as
the normal stimulated salivary flow group (25, 26).
Persons with a history of major salivary gland pathology
or extirpation were excluded from the analyses.
Data analysis
The data analyses were done by using SPSS 12Æ0** for
Windows. Difference in candidal activity, which was the
pH in the selective medium, was evaluated by the
*DENTSPLY – Senkin K. K., Tokyo.
**SPSS Inc., Chicago, IL, USA.
38 K . I K E B E et al.
Mann–Whitney U-test or the Kruskal–Wallis test
because the distributions did not have normality.
P-values £0Æ05 were considered to be statistically signi-
ficant. Pairwise comparisons after the Kruskal–Wallis
tests were made by means of the Mann–Whitney U-tests
with Bonferroni adjustments. Results were considered
significant at a ¼ 0Æ01 for three categories (27).
In addition, Spearman’s correlation coefficients were
used to evaluate the relationship between candidal
activity of tissue fitting surface of a maxillary denture
and palatal mucosa in the same individuals.
A multiple logistic regression analysis was used to
determine if an independent variable was statistically
significant after controlling for other variables. The
dependent variable was the proportion of the subjects
with high candidal activity of palatal mucosa (less than
the 25th percentile in pH of the medium) (28). All
independent variables evaluated were entered into the
model. Age and stimulated salivary flow rate were
continuous independent variables. Other independent
variables, such as gender, drugs intake and dental status
of maxilla, were transformed into dichotomous varia-
bles.
Results
Distribution of the pH in the selective medium after
incubation was skewed (Fig. 1). The mean pH of the
medium was 5Æ49 (s.d.: 0Æ37) and the median pH was
5Æ66.
There was a significant difference in the candidal
activity according to the dental status of the maxilla
(P < 0Æ001) (Table 1). The complete denture group
(n ¼ 38) showed significantly higher candidal activity
than the other two groups, and the removable partial
denture group (n ¼ 101) showed significantly higher
candidal activity than the natural dentition group
(n ¼ 212) in multiple comparisons (P < 0Æ01). Candidal
activity was likely to be higher in the hyposalivation
group (<0Æ5 mL min)1) than in the normal stimulated
salivary flow group (P ¼ 0Æ059). Stimulated salivary
flow rate was not significantly associated with dental
status (P ¼ 0Æ40) (Table 2).
In maxillary denture wearers, candidal activity of pal-
atal mucosa had a significantly positive correlation with
candidal activity of tissue fitting surfaces of a maxillary
denture (r ¼ 0Æ806, P < 0Æ001) (Fig. 2).
A multiple logistic regression analysis showed that
high candidal activity of the palate was significantly
ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 36–42
160
140
120
Number of persons
100
80
60
40
20
0
–4·25 –4·75 –5·25 –5·75 –6·25
–4·5 –5 –5·5 –6
pH of StomastatTM of palatal mucosa
Fig. 1. Distribution of candidal activity of palatal mucosa.
associated with being male and wearing maxillary
removable dentures (Table 2). The odds ratio was the
highest for dental status of maxilla (2Æ0 for RPD/natural
dentition, 3Æ9 for CD/natural dentition). Stimulated
salivary flow rate was likely to be negatively related to
high candidal activity (odds ratio ¼ 0Æ74, P ¼ 0Æ07)
(Table 3).
Discussion
Candidal parasitism to the palatal mucosa is usually
symptomless and may not always lead to stomatitis or
the need for treatment. However, decline in the body’s
defenses or drug therapy may predispose an increase
in numbers of Candida species or the transformation
of Candida species into a pathological state (2). Most
healthy elderly people inevitably become frail because
of disease or from the natural process of ageing and
they commonly wear removable dentures. Therefore, it
is important both for individuals and for public health
in general that information regarding risk factors of oral
infection and disease prevention be made available. For
this reason, we used persons who were symptom-free
of stomatitis as study subjects.
A number of epidemiological studies have yielded
information about the prevalence of denture-related
stomatitis (8, 9, 11, 29). However, there are remarkable
differences in the findings. These might be explained by
demographic variations, by the use of different diag-
nostic criteria, or by a lack of standardized definitions in
clinical examinations (30).
In examining colonized yeasts, the commonly used
method of conventional vital counting has several
 ASSOCIATION OF CANDIDAL ACTIVITY WITH DENTURE AND SALIVA 39

Table 1. Association of gender, age,

% of
drug intake, dental status and saliv-
subjects pH of StomastatTM
ary flow with candidal activity of
palatal mucosa Interquartile
Variables n % Mean s.d. Median range P-value*

Gender
Male 189 53Æ8 5Æ48 0Æ37 5Æ66 0Æ48 0Æ158
Female 162 46Æ2 5Æ52 0Æ35 5Æ68 0Æ36
Age
60–64 years 121 34Æ5 5Æ54 0Æ35 5Æ68 0Æ35 0Æ414
65–69 years 141 40Æ2 5Æ50 0Æ36 5Æ66 0Æ38
70 years and more 89 25Æ4 5Æ44 0Æ36 5Æ57 0Æ55
Medication
No 178 50Æ7 5Æ50 0Æ36 5Æ67 0Æ46 0Æ798
Yes 117 33Æ3 5Æ51 0Æ35 5Æ65 0Æ39
Dental status of maxillae
Complete denture 38 10Æ8 5Æ27 0Æ46 5Æ47 0Æ80 <0Æ001
Removable partial denture 101 28Æ8 5Æ44 0Æ37 5Æ54 0Æ51
Natural dentition 212 60Æ4 5Æ57 0Æ32 5Æ70 0Æ24
Stimulated salivary flow
less than 0Æ5 mL min)1 86 24Æ5 5Æ51 0Æ36 5Æ59 0Æ47 0Æ059
0Æ5 mL min)1 and more 265 75Æ5 5Æ45 0Æ36 5Æ67 0Æ42
Total 351 5Æ50 0Æ36 5Æ66 0Æ41

*Mann–Whitney U-test or Kruskal–Wallis test.

6·5 Table 2. Salivary flow rate by status of maxillae

n Mean s.d. P-value*

6·0
pH of StomastatTM of palatal mucosa

complete denture 38 0Æ97 0Æ76 0Æ403

removable partial denture 101 1Æ13 0Æ78
5·5
natural dentition 212 1Æ16 0Æ86

5·0 Total 351 1Æ13 0Æ83

4·5
4·0
3·5
3·5 4·0
pH of StomastatTM of denture surface
Fig. 2. Relationship between candidal activity of tissue fitting
surface of denture and palatal mucosa.
shortcomings, particularly in respect to its precision.
In order to objectively evaluate candidal activity, a
simple method of measuring Candida biofilms activity
using pH change in the liquid selective medium was
used in this study (23). The logarithm of the initial
*Kruskal–Wallis test.
y = 0·757x+1·39 number of yeasts from the inoculated swab was
r = 0·806, P < 0·001 linearly correlated with the pH value of StomastatTM
after both 24-h incubation periods (r ¼ 0Æ992,
P < 0Æ01), which confirmed the validity of this method
4·5 5·0 5·5 6·0 6·5 (31). This simple quantitative method could be
extended to the determination of the fungal levels
in denture or oral carriage in vivo. Therefore, this
method is quantitative, simple and suitable for large
fieldwork populations.
It has generally been assumed that old age may also
represent a predisposing condition for increased cand-
idal colonization. Lockhart et al. (32) demonstrated that
the frequency and intensity of carriage of candidal
colonization increased as a function of age, independ-
ent of denture use. However, our study demonstrated

ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 36–42

40 K . I K E B E et al.
B s.e. P-value
Age )0Æ043 0Æ034 0Æ200
Gender 0Æ601 0Æ293 0Æ040
Medication )0Æ181 0Æ266 0Æ497
Dental status (P-value ¼ 0Æ002)
CD 1Æ362 0Æ424 0Æ001
RPD 0Æ674 0Æ293 0Æ021
Salivary flow )0Æ311 0Æ174 0Æ074
RPD, removable partial denture; CD, complete denture.
Dependent variable: candidal activity of palate.
Age (years): continuous variable.
Gender: males ¼ 0, females ¼ 1.
Medication: No ¼ 0, Yes ¼ 1.
Dental Status: natural dentition was the reference category (¼0) for RPD or CD (¼1).
Salivary flow (mL min)1): continuous variable.
that candidal activity was not significantly associated
with age or gender in the relatively healthy old people.
This discrepancy may be caused by the difference in
mean age (77 versus 67 years) and chronic medical
conditions between the study samples.
The commonest form of oral candidosis is Candida-
associated denture stomatitis (9, 33). The maxillary
denture was reported as the major source of candidal
infection in long-term hospital care (9). High oral yeast
counts and frequent prevalence of oral candidosis in
elderly subjects living in institutions were associated
with poor oral hygiene and neglect of denture care
(11).
Our results indicated that using a maxillary denture
was the only significant independent variable associ-
ated with candidal activity, even in relatively healthy
older adults, after controlling for age, gender and drug
intake. This finding is not new: using imprint cultures,
the frequency of isolation of Candida albicans in com-
plete denture wearers was more than twice that of
dentate patients (34). Dentures may be the direct cause
of many oral mucosal lesions because of a change in the
oral cavity and a loading of the oral mucosal (30). In
addition, candidal activity of tissue fitting surfaces of a
maxillary denture is strongly associated with candidal
activity of the palatal mucosa, suggesting that wearing
dentures, especially maxillary complete dentures, plays
a role in creating a reservoir for disseminated infections
(35). Therefore, in order to treat oral candidosis, we
should disinfect or replace the dentures at the initial
stage of treatment. Surface irregularities of the denture
would increase the likelihood of microorganisms
remaining on the surface after the prosthesis is cleaned,
allowing continuous reinfection of the plate (36, 37), so
Table 3. Logistic regression analysis
Odds ratio 95% CI
for the candidal activity of palate
0Æ96 0Æ90–1Æ02 (n ¼ 351)
1Æ82 1Æ03–3Æ24
0Æ83 0Æ50–1Æ41
3Æ90 1Æ70–8Æ96
1Æ96 1Æ10–3Æ48
0Æ73 0Æ52–1Æ03
the denture surface must be polished as smooth as
possible.
Another possible reason for a change in the oral
cavity is hyposalivation. Narhi et al. (38) demonstrated
that subjects with low stimulated salivary flow rates
had significantly higher yeast counts than did subjects
with normal stimulated salivary flow rates in a group of
368 Finns aged 76, 81 and 86 years. Our results
indicated that persons with low stimulated salivary
flow tended to have high candidal activity in symptom-
free older adults.
Saliva mechanically cleans the oral cavity and redu-
ces the adhesion of C. albicans to the acrylic resin of
dentures (39, 40). Many salivary proteins protect the
soft tissue of the oral cavity (41). Human saliva contains
yeast inhibitors such as histatin, defensin or secretory
immunoglobulin A (42–45). Our finding reconfirmed
the importance of saliva in protecting the soft tissue of
the oral cavity, even in the relatively healthy older
adults.
This study suggests that the activity of Candida species
in the oral cavity is associated with the wearing of
removable dentures and stimulated salivary flow,
independent of age or gender even in the relatively
healthy elderly. The results emphasize the critical need
to measure candidal activity of denture wearers as
candidosis is usually symptomless and hyposalivation is
very common.
Acknowledgments
The authors greatly appreciated the grammatical cor-
rection of the manuscript by Joanne Madsen, M.A.
We are especially grateful to Ronald L. Ettinger BDS,
ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 36–42
 ASSOCIATION OF CANDIDAL ACTIVITY WITH DENTURE AND SALIVA
MDS, DDSc, Professor of University of Iowa, College of
Dentistry and Robert P. Renner DDS, Professor of State
University of New York at Stony Brook, School of
Dental Medicine for commenting and advising on the
manuscript. This investigation was partially supported
by a Grant-in-Aid for Scientific Research (No.
16390555) from Japan Society for the Promotion of
Science.
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Correspondence: Kazunori Ikebe, Division of Oromaxillofacial Regen-
eration, Osaka University Graduate School of Dentistry, University of
Osaka Faculty of Dentistry, 1-8 Yamadaoka Suita Osaka 565-0871,
Japan.
E-mail: ikebe@dent.osaka-u.ac.jp