Mycopathologia (2010) 169:365–372
DOI 10.1007/s11046-010-9275-8
Determining Candida spp. Incidence in Denture Wearers
Ozlem Abaci • Alev Haliki-Uztan •
Berran Ozturk • Suna Toksavul •
Mubin Ulusoy • Hayal Boyacioglu
Received: 22 June 2009 / Accepted: 20 January 2010 / Published online: 9 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The aim of this study was to determine
Candida spp. incidence in the oral cavity of denture
wearers and characterize predisposing factors in
denture-related stomatitis (DRS). Three groups of
denture wearers and a control group were evaluated
for DRS according to Newton’s classification. The
amount of yeast in saliva and the presence of yeast on
mucosal surfaces were determined by phenotyping
methods, and the impact of some risk factors on
candidal carriage was evaluated. The development of
DRS is most common in complete prosthesis users.
When the count of yeast in saliva is C400 cfu/ml, the
frequency of DRS is increased. In individuals who
develop DRS, the most frequently encountered spe-
cies that was identified as C. albicans. Prosthetic
hygiene was related to the intensity of candidal
growth and the development of DRS. C. albicans live
as saprophyte in the oral cavity. But, it is capable of
causing infection if there are predisposing conditions
O. Abaci (&)
A. Haliki-Uztan
Faculty of Science, Department of Biology,
Basic & Industrial Microbiology Section, Ege University,
Bornova, Turkey
e-mail: ozlemabaci@yahoo.com
B. Ozturk
S. Toksavul
M. Ulusoy
Faculty of Dentistry, Department of Prosthetic Dentistry,
Ege University, Bornova, Turkey
H. Boyacioglu
Faculty of Science, Department of Statistics,
Ege University, 35100 Bornova, Izmir, Turkey
related to the host. Usage of removable prosthesis may
cause these microorganisms to gain pathogenicity.
Keyword Candida albicans
Denture wearers

Denture-related stomatitis
Introduction
Long-term use of prosthesis is the most important risk
factor for Candida species colonization of the
mucosal surfaces, and this may be sufficient for the
development of oral candidiasis. Oral fungal infec-
tion that causes Candida-associated prosthesis sto-
matitis is a common disease in 50–60% of denture
wearers [1–3]. Candida spp. have the ability of
changing into an effective pathogen with factors such
as adhesion, dimorphism, enzymes (proteinases and
phospholipases), and biofilm formation. It has been
shown that adhesion of the mycelium form of
C. albicans to epithelial buccal cells is stronger,
and also proteinases secreted by mycelial form cause
damage in oral mucosa [4].
In patients using dental prosthesis, 400 cfu/ml in
saliva and higher yeast count often reveal Candida-
associated prosthesis stomatitis [5]. Besides this, in
individuals using dental prosthesis, different oral
infections such as chronical hyperplastic candidia-
sis (CHC) may develop. It is known that certain
C. albicans strains have the ability of producing
carcinogenic nitrosamines from saliva and promoting
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neoplastic changes [6]. Therefore, our primary pur-
pose in this study was to compare Candida spp.
incidence of the oral cavity in the individuals who
wore different types of dental prosthesis and that in
the individuals who did not wear one. We also aimed
to determine the species of yeasts, to evaluate the
mucosa in terms of prosthesis stomatitis, to evaluate
the impact of the age of the current prosthesis,
patients’ habit of using the prosthesis and their
awareness on prosthesis hygiene, genders of the
patients on candidal carriage [7].
Materials and Methods
Dental Status
Subjects were classified into four categories accord-
ing to the dental status of their maxilla: complete
denture, removable partial denture, fixed prothesis,
and natural dentition [2, 5, 8].
Patients
Thirty patients wearing a complete denture, 30 patients
wearing a removable partial denture and 30 patients
wearing a fixed partial denture and being treated for
prosthodontic treatment in Ege University, Dental
Faculty were included in our study for 12 months. The
control group consisted of 20 patients who had not
been treated for any denture-related problems.
Inclusion Criteria
Age played an important role for the inclusion criteria
of the patients. Those under the age 20 and over 80
were excluded. Moreover, those having systemic
disorders such as cardiovascular diseases, diabetes
mellitus, kidney problems, rheumatisms, and blood
diseases were excluded. Those who had been using
their dentures for at least 6 months and those who had
not taken any antibiotics and/or steroids at least for
the last month were included in the study.
Clinical Examination
Clinical examination was performed by the clini-
cians, who classified DRS in types I, II, III according
to the criteria proposed by Newton. Type I DRS
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Mycopathologia (2010) 169:365–372
showed localized inflammation or pinpoint hyper-
emia, type II showed a generalized erythema and type
III showed papillary hyperplasia of the palate [9].
Evaluation of the Dental Prosthesis
Evaluation of the dental prosthesis was made by
direct examination. The data analyzed included were
as follows: denture hygiene was classified as 0
(absence of plaque), 1 (presence of plaque), age of
the prosthesis and whether continuously or discon-
tinuously used was also noted.
Each participant was requested to fill in a ques-
tionnaire, which contains questions about their age,
gender, alcohol use, nutrition habits, general health
status, oral hygiene procedures, oral health, duration
the dentures used and denture wearing habits of
the participant. Oral health status of all individuals
was evaluated by the faculty members of the Ege
University, Faculty of Dentistry, Department of
Prosthetic Dentistry.
Samples
Total unstimulated saliva was collected during 5 min.
To standardize the effect of diurnal variations, meals
and tooth brushing, sampling was carried out at
midmorning at least 2 h after eating, drinking, or any
oral hygiene procedure. After salivary flow measure-
ment, saliva was sampled in Sabouraud Dextrose
Agar containing chloramphenicol (1 mg/ml), CHRO-
Magar Candida Medium (Becton–Dickinson Com-
pany, Sparks, MD).
Oral swabs were taken from palatal mucosa and
dorsum of the tongue by passing a sterile cotton swab
several times across the surface. Swabs were used to
inoculate CHROMagar Candida medium, which was
subsequently incubated for 48 h at 37°C.
Yeast colonies were counted after 48 h, and the
patients were classified according to the number of
cfu as follows: negative (cfu/ml: 0), carrier (cfu/ml:
\400), and positive (cfu/ml C400) [5]. One colony
of each morphological type on CHROMagar Candida
medium was plated for purity and stored. Candida
species isolated from saliva, palatal mucosa and
tongue were identified by germ tube and chlamy-
dospores production, and commercially available API
20C AUX yeast identification system (BioMerieux,
France) [10].
Mycopathologia (2010) 169:365–372 367

Salivary Properties
Subjects were classified into two groups according to
stimulated salivary flow rates. Persons whose unstim-
ulated flow rate was \0.3 ml min-1 were placed in
the hyposalivation group, and the rest were desig-
nated as the normal stimulated salivary flow group
[3]. Saliva samples were analyzed for pH by using pH
paper (Merck, pH: 0–14) [11].
Statistical Analyses
Statistical analysis was carried out using SPSS for
Windows 10.0 (SPSS Inc.; Chicago, IL).
Cross-tabulations were prepared. Chi-square anal-
ysis was performed to examine whether there was a
difference between groups. The Independent Samples
T Test was used to compare the mean value of two
groups. P values less than 0.05 were considered
statistically significant [12].
denture, C0.3 ml/min for 21 individual and \0.3 ml/
min for 9 individuals were determined. In fixed
partial denture users, C0.3 ml/min for 27 individuals
and\3 ml/min for 3 individuals were determined. pH
values were determined as 6–8 for all individuals.
Prevalence of DRS and Prosthesis Status
After the clinical examination, 18 of the 30 individ-
uals (60%) with complete prosthesis, 11 of the 30
individuals (36,7%) with removable partial denture
and 5 of the 30 with fixed partial prosthesis (16.7%)
developed prosthesis stomatitis. However, in the
control group which consisted of 20 individuals with
natural teeth, all the individuals were considered to
have healthy mucosa. Twenty of them experienced
type I prosthesis stomatitis and fourteen of them
experienced type II prosthesis stomatitis. When DRS
development and prosthesis types were compared in
these patients, the difference was statistically signif-
icant (P = 0.003 \ 0.05).

Results Quantitative Analysis

In our study, individuals who wore three types of
dental prosthesis and individuals who had natural
teeth were evaluated in terms of candidal carriage,
and the effects of the local factors on this carriage
were investigated. Distribution of ninety patients and
twenty controls according to gender and age is given
in Table 1.
Salivary Properties
Flow rate of saliva was evaluated as C0.3 ml/min for
28 individuals in complete denture users and
\0.3 ml/min for 2 individuals. For removable partial
While no yeast reproduced in the saliva samples of 49
of the individuals with healthy mucosa, there was
yeast reproduction in the saliva samples of five of
them. Whereas the number of yeast cells in the saliva
samples of 17 of the individuals with healthy mucosa
was also \400 cfu/ml, the number in eleven individ-
uals who developed DRS was \400 cfu/ml. Whereas
the number of yeast cells in the saliva samples of nine
of the individuals with healthy mucosa was ‡400 cfu/
ml, it was ‡400 cfu/ml in the 19 individuals who
developed DRS. As a result, when the number of yeast
cells in the saliva is C400 cfu/ml, frequency of DRS
development increases (P = 0.00 \ 0.05) (Table 2).

Table 1 Distribution of 90 patients and 20 controls according to gender and age

\41 41–50 51–60 61–70 [70 T
CD RPD FPD ND CD RPD FPD ND CD RPD FPD ND CD RPD FPD ND CD RPD FPD ND

M 0 0 3 6 0 3 6 0 6 9 1 0 1 4 1 0 6 0 1 0 47
F 0 0 4 14 2 3 6 0 6 9 6 0 6 2 2 0 3 0 0 0 63
T 0 0 7 20 2 6 12 0 12 18 7 0 7 6 3 0 9 0 1 0 110
M Male, F Female, T Total
CD Complete denture, RPD Removable partial denture, FPD Fixed partial denture, ND Natural dentition

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Table 2 Candida species
Candida counts in
counts in saliva of 90 denture
saliva (cfu/ml)
wearers and 20 control
subject 0 49 (90.7%)
\400
C400
Total
Colonization of Palatal Mucosa by C. albicans
When DRS development and palatal C. albicans
colonization were evaluated, the frequency of isola-
tion of C. albicans was 10.53% in individuals with
healthy mucosa, it was 67.65% in those who expe-
rienced prosthesis stomatitis. No yeast cells repro-
duced in 84.21% of individuals with healthy mucosa.
While non-C. albicans reproduced in 14.71% of the
individuals who suffered from prosthesis stomatitis,
in 2.94% of them, in addition to C. albicans, another
yeast species reproduced (P = 0.00 \ 0.05).
Distribution of Candida spp. Identified
The Table 3 shows yeast species isolated from saliva,
palatal mucosa, and tongue mucosa samples taken
from the individuals using different types of prosthe-
ses and from those of the control group (Table 3).
Dental Hygiene and Using Habit
When the individuals using complete denture and
those using removable partial denture were evaluated
together, it was considered that prosthetic hygiene
was related to the intensity of candidal growth
(P = 0.00 \ 0.05) and the development of DRS
(P = 0.002 \ 0.05). On the other hand, when the
individuals wearing dentures (complete and partial)
were evaluated together, the frequency of denture
cleaning was not related either to the density of
candidal growth (P = 0.872 [ 0.05) or the develop-
ment of DRS (P = 0.6 [ 0.05). And, also the using
habit of the dentures were not related to density of
candidal growth (P = 0.983 [ 0.05) and develop-
ment of DRS (P = 0.9 [ 0.05) (Tables 4 and 5).
Gender
DRS development was observed in 8 males and 10
females wearing complete dentures, in 6 males and 5
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Mycopathologia (2010) 169:365–372
Healthy mucosa DRS (Denture-related Total
stomatitis)
5 (9.3%) 54
17 (60.7%) 11 (39.3%) 28
9 (32.1%) 19 (67.9%) 28
75 (68.2%) 35 (31.8%) 110
females wearing removable partial dentures and only
in 4 males wearing fixed partial dentures. In 30 female
and 20 male individuals, Candida was not determined
in saliva samples. In 19 female and 12 male partic-
ipants, Candida counts in saliva was determined as
\400 cfu/ml. Over 400 cfu/ml, Candida spp. was
found in the saliva of 15 female and 14 male patients.
As a result, the relationship between the development
of DRS in palatal mucosa and gender (P =
0.628 [ 0.05), and between the number of yeast cells
in the saliva and gender (P = 0.746 [ 0.05) was not
considered statistically significant.
Age of the Prosthesis
The relationship between the age of the prosthesis,
the number of the colonies (P = 0.465 [ 0.05), and
also development of DRS (P = 0.266 [ 0.05) were
not statistically significant.
Discussion
C. albicans is microorganism existing in the oral flora
of healthy individuals with or without teeth. There are
several factors leading to the presence of C. albicans in
oral cavities, their increase in number and gaining
pathogenicity. Some of these factors are diabetes
mellitus, AIDS, long-term antibiotic or corticosteroid
use, due to head–neck cancer radiation therapy imple-
mentation, or nutritional disorders. It is also known that
using prosthesis candidal colonization increases and
thus leads to denture-related stomatitis resulting from
Candida. Saliva prevents adhesion of C. albicans to
acrylic surfaces; however, if serum enters oral cavities
as a result of trauma in palatal mucosa, this increases
the chance of adhesion. Proteinase secreted by the
filamentous form by the organism may cause damage
in the oral mucosa. Therefore, the density of Candida
in individuals using prosthesis is important in terms of
infection development, and the determination of the
 Table 3 Candida species in (3a) saliva, (3b) on dorsum of the tongue and (3c) palatal mucosa
Complete denture (n:30) Removable partial denture (n.30) Fixed partial denture (n:30) Natural dentition (n:20)
Without DRS Without DRS Without DRS Without With
DRS (n:12) (n:18) DRS (n:19) (n:11) DRS (n:25) (n:5) DRS (n:20) DRS
Mycopathologia (2010) 169:365–372

(3a) Candida species in saliva

0 9 (75%) 0 8 (42.1%) 0 16 (64.0%) 2 (40%) 13 (65%)
C. albicans 2 (16.7%) 12 (66.7%) 8 (42.1%) 8 (72.7%) 7 (28.0%) 1 (20%) 7 (35%)
C. glabrata 1(5.6%) 1 (5.3%)
C. kefyr 2 (10.5%) 1 (20%)
C. krusei 1 (20%)
C. tropicalis 1 (8.3%)
C. sphaerica 1 (9.1%)
C. famata
C. albicans ? C. glabrata 2 (11.1%) 2 ((18.2%)
C. albicans ? C. kefyr ? C. krusei 1 (5.6%)
C. albicans ? C.krusei 1 (5.6%)
C. albicans ? C. krusei ? S. cerevisiae ? C. tropicalis 1 (5.6%)
C. keyfr ? C. famata 1 (4%)
C. keyfr ? C. krusei 1 (4%)
(3b) Candida species on dorsum of the tongue
0 12 (100%) 4 (22.2%) 13 (68.4%) 2 (18.2%) 18 (72%) 3 (60%) 18 (90%)
C. albicans 8 (44.4%) 5 (26.3%) 7 (63.6%) 5 (20%) 1 (20%) 2 (10%)
C. glabrata 3 (16.7%) 1 (9.1%)
C. kefyr 1 (5.3%) 1 (4%)
C. krusei 1 (5.6%) 1(20%)
C. sphaerica 1 (9.1%)
C. famata 1 (4%)
C. albicans ? C. krusei 1 (5.6%)
C. tropicalis ? S. cerevisiae 1 (5.6%)
369

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Complete denture (n:30) Removable partial denture (n.30) Fixed partial denture (n:30) Natural dentition (n:20) Table 4 The relationship of density of candidal growth to

With
DRS
denture hygiene and to frequency of denture cleaning and to
prosthesis using habits
Candida counts in saliva
DRS (n:20)

19 (95%)
1 (5%)
Without

Denture hygiene status 0 \400 C400 Total

Absence of the plaque 13 1 4 18
Presence of the plaque 8 15 19 42
3 (60%)
1 (20%)

1 (20%)
Frequency of denture cleaning
(n:5)
DRS

Irregular – 3 3 6
One time per day 10 4 9 23
C2 times per day 11 9 11 31
DRS (n:25)

19 (76%)
4 (16%)

1 (4%)

1 (4%)
Without

Using habit
Removing 10 7 11 28
Not removing 9 7 11 27
Sometimes 2 2 1 5
1 (9.1%)
8 (72.7%)
2 (18.2%)
(n:11)
DRS

Table 5 The relationship of development of DRS to denture

0

hygiene and to frequency of denture cleansing and to prosthesis

using habits
DRS (n:19)

DRS (Denture-related stomatitis)

Without

1 (5.6%) 14 (73.7%)
3 (15.8%)
1 (5.3%)
1 (5.3%)

Without DRS With DRS Total

Denture hygiene status

Absence of the plaque 15 3 18
1 (5.6%)
1 (5.6%)

1 (5.6%)
1 (8.3%) 14 (77.8%)

Presence of the plaque 16 26 42

DRS (n:12) (n:18)
DRS

Frequency of denture cleaning

Irregular 1 5 6
One time per day 14 9 23
Without

11 (91.7%)

C2 times per day 16 15 31

Using habit
Removing 14 14 28
Not removing 14 13 27
Sometimes 3 2 5
(3c) Candida species on the palatal mucosa

species that cause the infection, is of importance in

order to start the proper treatment [11, 13].
The oral cavity is a reservoir for colonization and
C. albicans ? C. tropicalis

infection of systemic organs by pathogenic microor-

ganisms. C. albicans has been isolated from transtrac-
heal aspirates from patients with pleuropulmonary
Table 3 continued

infection and other pulmonary samples. The associa-

tion seems to occur only in patients with a severely
C. sphaerica
C. glabrata
C. albicans

C. famata

compromised health, in frail elderly people and in

C. krusei
C. kefyr

patients with chronic pulmonary diseases. The coex-

istence of Pseudomonas spp. and C. albicans in elderly
0

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Mycopathologia (2010) 169:365–372
people is a potential indicator of high risk for
pneumonia and heart disease [14, 15].
Evidence is indeed increasing that oral infections
may also play a role in the pathogenesis of many
systemic diseases. Oral infections may affect the
general prognosis of not only the old, fragile and
immunocompromised, but also of those who are
healthy [14, 15].
However, there are not enough studies investigat-
ing the effects of the types of the prosthesis on
colonization. Therefore, we compared candidal car-
riage in the individuals using these three different
types of prostheses and the individuals with their
natural teeth. In order to refrain from obtaining any
misleading results, we included in our study only
those individuals who had no systemic disorders, or
those who were not taking corticosteroids or immu-
nosuppressive medicines.
It is reported that development of DRS, in
individuals who used their prosthesis properly, ranged
from 11 to 67% [14]. In our study, DRS developed in
60% of the individuals with total prosthesis, 36.7% of
the partial prosthesis users and 16.7% of the fixed
prosthesis users. Development of DRS is less severe
in the partial prosthesis users than in the total
prosthesis users and mild in fixed prosthesis users.
This might be due to the material used in prosthesis.
That is because adhesion of C. albicans to acrylic
surfaces used in total prostheses and to mucosa is
more than to other surfaces [16–18]. And, in fixed
prosthesis, most do not contact the mucosa and most
do not contain denture material. The findings of our
study also show that the difference is significant when
the development of DRS and the prosthesis types are
compared (P = 0.003 \ 0.05).
Statistical analysis according to Newton types had
shown significant relation between category of sto-
matitis and number of detected yeast colonies in saliva
[5, 19]. It is known that oral concentration of Candida
species in individuals, who suffer from DRS is higher
than those who do not [5, 6]. Yeast count of C400 cfu/
ml in prosthesis user points out Candida-related
denture stomatitis [5]. Our results also revealed that
the frequency of DRS development is higher in
individuals with yeast count of ‡400 cfu/ml, and the
result is statistically significant (Table 2). We also
demonstrated that the presence of C. albicans was
more frequent in denture-related stomatitis. The
results we obtained showed that there were 77.8% of
371
C. albicans, 5.6% of C. glabrata, 5.6% of C. kefyr,
and 5.6% of C. albicans and C. tropicalis were also
determined in the palatal smear samples of the
individuals who used complete prosthesis and devel-
oped DRS. When the saliva samples of the individuals
who used complete prosthesis and developed DRS
were investigated, it was also found that at a rate of
66.7% of C. albicans is a common, then 5.6% of
C. glabrata, 11.1% of C. albicans and C. glabrata,
5.6% C. albicans, C. kefyr, and C. krusei, 5.6% of
C. albicans and C. krusei and 5.6% of C. albicans,
C. krusei, S. cerevisiae and C. tropicalis were
determined. On their tongues, 44.4% of the colonies
were determined as C. albicans, 16.7% at C. glabrata,
5.6% at C. krusei, 5.6% at C. albicans and C. krusei,
5.6% at C. tropicalis and S. cerevisiae were also
present. The rate of C. albicans was 72.7% on the
palatal mucosa of the individuals who used partial
prosthesis and developed DRS. The rate of C. glabrata
in those individuals was 18.2%, which was followed by
9.1% of C. sphaerica. The rate of C. albicans was
63.6% on the tongue of them, while the rate of
C. glabrata was 9.1% which was followed by 9.1% of
C. sphaerica (Table 3). Since the most widespread
species in the saliva, palatal mucosa, and tongue
mucosa samples of the individuals who developed
DRS was C. albicans, this suggests that the major cause
of prosthetic stomatitis or DRS development is
C. albicans. It is generally acknowledged that the
presence of the opportunistic C. albicans in denture
plaque is considered as an important factor in the
development of this inflammation [16, 20].
It is also stated that prosthesis stomatitis is mostly
associated with the presence of high counts of
Candida spp. in the saliva and poor prosthetic
hygiene [3]. In our study, the relationship between
presence of plaque and density of candidal growth in
saliva (P = 0.00 \ 0.05) and also denture stomatitis
was statistically significant (P = 0.002 \ 0.05). This
result is in accordance with the results of the study,
which demonstrated a higher frequency of denture
stomatitis in patients who do not cleaning their
dentures properly [3]. However, the relationship of
density of candidal growth in saliva and development
of DRS to frequency of denture cleaning was not
statistically significant (Tables 4 and 5).
Continuous denture wearing appears to facilitate
denture stomatitis by increasing the local injury and
the time of mucosal exposure to denture plaque.
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Sadomari et al. [21] stated that edentulous patients
wearing dentures during sleep showed much more
occurrence of denture stomatitis than those who do
not have that habit. However, according to results of
our study, the relationship between overnight denture
wearing and density of the candidal growth in saliva
and also denture stomatitis was not statistically
significant (Tables 4 and 5).
Studies conducted on this topic report that devel-
opment of DRS in women is higher than in men due to
hormonal changes [18]. It was found that there was no
statistically significant difference in gender and the
amount of yeast (P = 0.746 [ 0.05) and also gender
and development of the DRS (P = 0.628 [ 0.05).
Moreover, we did not found a significant relationship
between denture stomatitis and increasing age of the
denture (P = 0.266 [ 0.05) and also amount of yeast
(P = 0.465 [ 0.05).
As a result, when all these findings were considered,
it was observed that C. albicans, which survive in the
oral cavity as saprophyte, increase in numbers and gain
pathogenicity after a prosthetic apparatus is used.
This study was approved by T.C Ege University
Ethical Committee of Medical Faculty with the serial
number 06–5.1/10 on June 20, 2006.
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