Journal of Advances in Medicine and Medical Research

27(6): 1-8, 2018; Article no.JAMMR.43719

ISSN: 2456-8899
(Past name: British Journal of Medicine and Medical Research, Past ISSN: 2231-0614,
NLM ID: 101570965)

A Comparative Study of Time Dependent Candidal

Colonisation in Diabetic and Non-diabetic Complete
Denture Patients
Mujtaba Ashraf1, Mariyam Ali1, Abhishek Gaur1, Monika Rajani1, A. K. Verma1,
Pratibha Katiyar1, Kaushik Kumar Pandey1* and Fauzia Tarannum1
1
Department of Prosthodontics, Career Post Graduate Institute of Dental Sciences, Lucknow, India.

Authors’ contributions

This work was carried out in collaboration between all authors. Authors Mujtaba Ashraf, Mariyam Ali
and AG designed the study, performed the statistical analysis, wrote the protocol and wrote the first
draft of the manuscript. Authors MR, AKV and PK managed the analyses of the study. Authors KKP
and FT managed the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JAMMR/2018/43719
Editor(s):
(1) Dr. Mohamed Essa, Department of Food Science and Nutrition, Sultan Qaboos University, Oman.
Reviewers:
(1) Adam Husein, University of Science, Malaysia.
(2) Renata Souto, Federal University of Rio de Janeiro, Brazil.
(3) Reginaldo dos Santos Pedroso, Federal University of Uberlândia, Brazil.
Complete Peer review History: http://www.sciencedomain.org/review-history/26206

Received 20 June 2018

Original Research Article Accepted 04 September 2018
Published 12 September 2018

ABSTRACT

Introduction: Acrylic dentures are important predisposing factors for oral candidiasis. Duration of
wearing dentures in the oral cavity of diabetic and non-diabetic patients can increase Candida
colonisation and results in the higher incidence of oral and systemic candidiasis.
Aims and Objective: 1. To compare the changes in Candidal colonisation with time in complete
denture prosthesis in diabetics and non-diabetics. 2. To evaluate the change in Candidal growth in
diabetics and non-diabetic complete denture wearers. 3. To evaluate the relation of time with
Candidal growth in complete denture wearers.
Materials and Methods: Three Salivary samples from each 20 edentulous, non-insulin dependent
diabetic and 20 edentulous (Group-1), non-diabetic subjects (Group-2) were collected at 3 different
time intervals. First sample was collected on the day of denture insertion, the second sample after
one week from the day of insertion and third sample, after one month from the day of denture
_____________________________________________________________________________________________________

*Corresponding author: E-mail: kaushik.pandey05@gmail.com;

 Ashraf et al.; JAMMR, 27(6): 1-8, 2018; Article no.JAMMR.43719

insertion in sterile containers using oral rinse technique. All samples were cultured directly on
Sabouraud agar medium and species identification was done by HiCrome Candida Differential Agar
culture methods.
Results: Candidal carriage and colonisation is more in diabetic denture wearer patients than non-
diabetic denture wearer patients. Candidal growth in complete denture wearers was significantly
increased from Day 1 to last day of 1st week (p=0.006), Day 1 to last day of 1st Month (p=0.005)
st st
and last day of 1 Week to last day of 1 Month (p=0.007) in Group 1. There was significant
mean change in Group 2 from last day of 1st Week to last day of 1st Month (p=0.001). The
common species of Candida isolated in both diabetic and non-diabetic denture wearer patients are:
C. albicans, C. glabrata and C. tropicalis.

Keywords: Candidal colonisation; complete dentures; diabetes mellitus.

1. INTRODUCTION 1.2 Objectives

Diabetes mellitus is a common and globally
growing health problem with a high morbidity and
mortality. The prevalence of diabetes is swiftly
increasing over the globe at an alarming rate.
According to the International Federation of
Diabetes, 415 million adults around the world are
suffering from diabetes, and it is estimated that
the numbers will reach around 642 million by
2040. India leads the World and stands at the
second position after China, with 69 million
persons affected by diabetes [1].
Diabetes mellitus is a chronic metabolic disease
that occurs when the body cannot produce
enough insulin, or cannot use insulin and is
characterised by constant increased glucose
levels in the blood (hyperglycemia) associated
with alterations in carbohydrate, protein and lipid
metabolism [2].
 To evaluate change in Candidal growth in
diabetics and non-diabetic complete
denture wearers.
 To evaluate the relation of time with
Candidal growth in complete denture
wearers.
2. MATERIALS AND METHODS
This prospective, in-vivo study was conducted in
the Department of Prosthodontics and Crown &
Bridge, Career Post Graduate Institute of Dental
Sciences and Hospital and Department of
Microbiology, Career Institute of Medical
Sciences and Hospital, Lucknow, after taking
informed consent from the patients. Approval
was obtained from the ethical committee of
Career Post Graduate Institute of Dental
Sciences and Hospital, Lucknow.

Oral candidiasis is an opportunistic fungal 2.1 Patient Selection

infection which is commonly associated with
Diabetes [3] all forms of oral candidiasis are
considered opportunistic and hence the epithet
‘the disease of the diseased’ has been given to
this condition [4].
Due to high rates of diabetes mellitus in the
elderly Indian population and to increase our
understanding of growth of Candidal colonies,
pathogenicity and related complication, this
research work was designed for a comparative
study of time dependent Candidal colonisation in
diabetic and non-diabetic complete denture
patients.
1.1 Aims
To compare the changes in Candidal
colonisation with time in complete denture
prosthesis in diabetics and non-diabetics.
Subjects included in this study were having
following inclusion criteria; completely edentulous
patients, diabetic and non-diabetic subjects aged
between 45-75 years. Exclusion criteria were;
patients with lost or broken dentures, HIV,
Immuno-compromised and highly debilitated
patients.
Forty new complete denture wearing patients
reporting to the Department of Prosthodontics,
fulfilling the inclusion criteria, which were
included in the study were divided into 2 groups:-
 Group 1: Edentulous, non-insulin
dependent diabetic subjects. (FBS>130
mg/dl)*
 Group 2: Edentulous, non-diabetic
subjects. (FBS<110 mg/dl)*
*FBS= Fasting Blood Sugar level

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 Ashraf et al.; JAMMR, 27(6): 1-8, 2018;; Article no.JAMMR.43719
no.

The diabetic status of the patients was
determined by the medical history of the previous
diagnosis of diabetes and their fasting blood
glucose levels were also determined before the
sample collection using colorimetric method. The
patients were assessed as non- diabetics on the
ground of fasting plasma glucose levels less than
110 mg/dl.
2.2 Fabrication of Dentures
All complete dentures included in this study was
fabricated, following standard balanced denture
protocol.
2.3 Sample Collection
Samples were collected by the concentrated oral
rinse technique as described by Samaranayake
et al. [5]. Subjects were instructed not to eat and
drink 2 hours prior to the sample collection. Each
individual was given 10 ml of sterile phosphate
buffer saline solution (0.1M solution with 7.2 pH)
and was asked to rinse mouth for 60 seconds
with this solution. Then that mouth rinse was
collected in sterile containers and was sent to the
microbiological laboratory.
Three samples were collected from each subject.
st
 1 sample: On the day of denture insertion
 2nd sample: After one week from the day of
insertion and
rd
 3 sample: After one month from the day
of denture insertion.
2.5 Microscopic Evaluation of Candidal
Colonies
All creamy, pasty colonies on SDA plates (Fig.
(Fig 1)
after 48 hrs were picked up and further
identification of Candidal species was done by
Hi-Crome
Crome Candida Differential Agar Agar. All
presumptive budding yeast like cells with pseudo
hyphae formation were identified as Candidal
species and were subcultured on Hi-Crome
Candida Differential Agar.
2.6 Culture on Candida Differential Agar
Small inoculums from isolated Candidal colonies
from Sabouraud’s Dextrose Agar were picked up
with a sterile inoculating loop, plated on Hi-
Crome Candida Differential Agar and were
incubated at 37°C for 24-48 hours (Fig. 2).
The different species of Candida were identified
based on the colour displayed by them on Hi-
Crome Candida Differential Agar [6
6,7,8].
C. albicans (CA) = light green
C. glabrata (CG)= = white pink, purple
C. tropicalis (CT) = dark blue to blue gray
C. gulliermondii= pale pink
C. kefyr= pink
C. krusei (CK) = pale pink, purple (rough with
spongy pale edges)
C. parapsilosis= white, pale pink

2.4 Inoculation and Incubation of

Samples into the Culture
ulture Media

Oral rinse samples were then inoculated onto

Sabouraud Dextrose Agar plates. The SDA
plates were prepared according to the
manufacturer’s instruction. The oral rinse sample
was vortex mixed prior to plating. A drop of oral
rinse sample was taken with 4 mm internal
diameter inoculating loop and the samples were
inoculated. In this study, 4 mm loop was taken to
standardise the quantity of samples. The plates
were then incubated aerobically for 48 hours at
37°C. The subject was designated as Candidal Fig. 1. Candidal culture on sabouraud
carrier if there was any growth of Candidal dextrose agar
colonies. A semiquantitative estimation of colony
count was done and growth was expressed as 2.7 Statistical Analysis
CFU/ml with dilution factor of 10 [3
3] for 0.001 ml
of inoculum. The Chi-square
square test was used to compare the
categorical variables. Unpaired t-test
test was used to
CFU/ml = Colony Forming Units / milliliters. compare normally distributed variables between

3
 Ashraf et al.; JAMMR, 27(6): 1-8, 2018;; Article no.JAMMR.43719
no.

the groups. Mann-Whitney

Whitney U test was used to significant mean change in Group 2 from
st st
compare non-normal
normal variables between the last day of 1 Week to last day of 1 Month
groups at different time periods.
ods. Wilcoxon rank (p=0.001) (Table 4).
sum test was used to compare the mean change 5. In Group 2 (n=20), 0 (0%), 2 (10%) and
in the continuous variables from Day 1 to 15 (75%) shows positive growth (p (p-
subsequent time periods. The p-value<0.05
value<0.05 was value1=0.001) on SDA sample 1, sample 2
considered significant. All the analysis was and sample 3 respectively.
carried out on SPSS 16.0 version. 6. The growth of various Candidal colonies
(CA+CG+CT+CK) was significantly
st
increased from Day 1 to last day of 1
week (p=0.001), Day 1 to last day of 1st
st
Month (p=0.0001) and last day of 1 Week
st
to last day of 1 Month (p=0.0001) in
Group 1. There was a significant mean
st
change in Group 2 from last day of 1
st
Week to last day of 1 Month (p=0.001)
(Table 5).

Table 1. The age distribution between the

groups

Groups Age in years (Mean±SD)

Group 1 58.45±6.43
Group 2 54.40±7.14
p-value 0.06
rome Candida
Fig. 2. Candidal culture on HiCrome
differential agar 4. DISCUSSION

3. RESULTS India leads the world with largest number of

1. The mean age of patients of Group 1 and
Group 2 was 58.45±6.43 and 54.40±7.14
years respectively (Table 1).
2. Candidal growth in complete denture
wearers was found to be significantly
(p=0.0001) higher in Group 1 compared to
st
Group 2 at Day 1, last day of 1 week and
st
last day of 1 Month (Table 2).
3. In Group 1 (n=20), 16 (80%), 19 (95%) and
20 (100%) shows positive growth (p-value
(p
1=0.001) on SDA sample 1, sample 2 and
sample 3 respectively (Table 3).
4. Candidal growth in complete denture
wearers was significantly increased from
st
Day 1 to last day of 1 week (p=0.006),
st
Day 1 to last day of 1 Month (p=0.005)
st st
and last day of 1 Week to last day of 1
Month (p=0.007) in Group 1. ThereT was
diabetic subjects earning the dubious distinction
of being termed the ‘diabetes capital of the world.
The intrusion of Western culture into the lives of
traditional indigenous communities has also had
devastating results in terms of the rise in
diabetes and related metabolic disorders [9].
[
Candida is an opportunistic pathogen, resulting
in disease when the host-commensal
commensal relationship
is disturbed [3]. Oral infection with Candida (a
yeast-like fungus) s) is a commoninfection,
especially in theelderly people [10].
]. The infection
caused by Candida sp. is associated with local
and systemic factors too. The use of a dental
prosthesis is indispensable for function and
esthetic rehabilitation of edentulous patients.
pat
The adhesion of microorganisms to a surface
is a prerequisite for the colonisation of that
surface [11].

Table 2. Comparison of Candidal growth in complete denture wearers (SDA) between the
groups across the time periods

Time period Group 1 Group 2 p-value

value
Day 1 40.20±30.57 0.00±0.00 -
Week 1 54.90±29.90 1.75±5.44 0.0001*
Month 1 60.40±30.85 15.40±14.91 0.0001*
Mann-Whitney U test, *Significant

4
 Ashraf et al.; JAMMR, 27(6): 1-8, 2018; Article no.JAMMR.43719

Table 3. Comparison of SDA growth between the groups

Group 1 (n=20) Group 2 (n=20) p-value

No. % No. %
Day 1
Growth 16 80.0 0 0.0 0.001*
No growth 4 20.0 20 100.0
Week 1
Growth 19 95.0 2 10.0 0.001*
No growth 1 5.0 18 90.0
Month 1
Growth 20 100.0 15 75.0 0.01*
No growth 0 0.0 5 25.0
Chi-square test, *Significant

Table 4. Comparison of mean change in Candidal growth in complete denture wearers (SDA)
from Day 1 to subsequent time periods in groups

Time period Group 1 Group 2

Mean change p-value Mean change p-value
Day 1 to Week 1 14.70±21.52 0.006* 1.75±5.44 -
Day 1 to Month 1 20.20±21.60 0.005* 15.40±14.91 -
Week 1 to Month 1 20.20±21.60 0.007* 13.65±15.25 0.001*
Wilcoxon Rank sum test, *Significant

Table 5. Comparison of growth of various Candidal colonies (CA+CG+CT+CK) between the

groups across the time periods

Time period Group 1 Group 2 p-value

Day 1 13.15±8.93 0.00±0.00 -
Week 1 18.35±8.36 1.25±4.55 0.0001*
Month 1 29.60±11.77 10.20±8.10 0.0001*
Mann-Whitney U test, *Significant

Table 6. Comparison of mean change in growth of various Candidal colonies (CA+CG+CT+CK)

from Day 1 to subsequent time periods in groups

Time period Group 1 Group 2

Mean change p-value Mean change p-value
Day 1 to Week 1 5.20±5.44 0.001* 1.25±4.55 -
Day 1 to Month 1 16.45±11.49 0.0001* 10.20±8.10 -
Week 1 to Month 1 11.25±8.73 0.0001* 8.95±6.65 0.001*
Wilcoxon Rank sum test, *Significant

There have been many studies on the adhesion
of Candidaal bicans to denture acrylic resin,
caused by the association of the commensal,
opportunist pathogen yeast with denture-induced
stomatitis. The presence of a denture in the oral
cavity, associated with the local alterations of the
oral mucosa and the systemic complications,
may render the denture wearer patient with
diabetes even more prone to Candidal infection.
A significantly higher incidence of Candida
infection and increased levels of Candida spp.
were found in diabetic patients wearing
removable dentures.
The results of the present study can be explained
on the basis of two factors:
a) Local: acrylic prosthesis
b) Systemic: diabetes.
Acrylic dentures act as a predisposing factor in
the occurrence of oral Candidal infection.
The dentures can act as a reservoir of infection
[12], because of the surface irregularities,
improper fit and suboptimal hygiene. The
collection of samples was done by Oral Rinse
Technique.

5
 Ashraf et al.; JAMMR, 27(6): 1-8, 2018; Article no.JAMMR.43719
The adhesion of Candida depends on the
microporosity present on the inner surface of the
denture. Substrate surface properties, such as
surface charge, surface free energy,
hydrophobicity and roughness of denture have all
been reported to influence the Candidal
colonisation. Acrylic resins are hydrophilic,
pervious and exhibit more water sorption. This
water sorption may help the Candida cells to
adhere or to even penetrate the surface of acrylic
resin. Surface irregularities of acrylic resin act as
a factor in the entrapment of microorganisms
[9,13].
The most frequently used primary isolation
medium for Candida is SDA [14] which, although
permitting growth of Candida, suppresses the
growth of many species of oral bacteria due to its
low pH. Typically SDA is incubated aerobically at
37°C for 24–48 hours. Candida develop as
cream, convex colonies on SDA and
differentiation between species is rarely
possible. It is estimated that more than one
Candida species occurs in approximately 10% of
oral samples and in recent years the ability to
detect non-albicans species has become
increasingly important. As a result, it has been
recommended that SDA should be used in
combination with a second differential medium
[15].
Hi-Crome agar was used as a second differential
medium for Candida sp. in this study. On Hi-
Crome agar, the differentiation is done on the
basis of strongly contrasted colony colors
produced by reactions of species specific
enzymes with a proprietary chromogenic
substrate. CHROMagar is recommended as a
useful isolation medium capable of the
presumptive identification of the yeast species
most commonly isolated from clinical material
and facilitating recognition of mixed yeast
cultures [6,7].
The Candida carriage rate in oral cavity was
found to be different in the various studies done
earlier. This could be due to the different
methods of sampling used, for example: oral
swabs, rinse using water or buffer. The oral rinse
technique with phosphate buffer solution was
used for sampling in the present study since this
is known to be a sensitive technique for
estimating the oral Candidal carriage. Also
CHROMagar shows 100% specificity and 100%
sensitivity when compared to Sabouraud
Dextrose Agar and conventional methods
[16,17,8,18,19,20].
6
In the present study, mean age of the Group
1(Non-insulin dependent diabetic subjects) and
Group 2 (Non-diabetic subjects) was 58.45±6.43
and 54.40±7.14 years respectively. There was no
significant (p>0.05) difference in the age
between the groups showing comparability of the
groups in terms of age.
In Group 1 and Group 2 (n=40) the prevalence of
Candida increases in Sample 1, Sample 2 and
sample 3, to 40%, 52.5% and 87.5%
respectively. Candida has affinity for the acrylic
surface of dentures and the organism can be
opportunistic, which can be explained by the fact
that dentures decrease the flow of oxygen and
saliva to the underlying tissue producing a local
acidic and anaerobic micro-environment that
favours yeast overgrowth [3].
In the present study, the number of CFU of
Candida is found to be more in diabetic denture
wearer subjects as compared to the non-diabetic
denture wearer. These results are similar to the
few previous studies done evaluating the same
factor [21]. Daniluk T et al. [22] had earlier
evaluated the occurrence rate of oral C. albicans
in denture wearer patients. The growth of various
Candida species on CA was found to be
significantly (p=0.0001) higher in Group 1
compared to Group 2 at Week 1 and Month 1.
Positive culture of Candida enrolled on the
denture of all diabetic subjects (100%) but this
was seen in (75%) of non-diabetics after 1 month
use of denture (p=0.01). Similar observation was
noted in a study by Kamran MHL [3] also
revealed that diabetes mellitus increased the
colonisation of Candida in denture and mouth.
The predisposition of the diabetics to infections
by pathogenic fungal species has been explained
in terms of enhancement of yeast growth by
elevated tissue fluid glucose levels. Moreover,
the presence of a high concentration of salivary
glucose combined with low salivary secretion
may enhance growth of yeasts and their
adherence in epithelial oral cells [12].
In the present study, the number of CFU of
Candida was found to be more in diabetic
subjects than the non-diabetic groups. These
results are similar to the report by Tapper-Jones
LM et al. [21] that stated the higher Candidal
density in diabetics. With multiple imprint culture
technique, they have found that 60% of the
diabetic group harboured C. albicans, which was
similar to the carrier rate determined by the
swabs and was considerably more than the value
 Ashraf et al.; JAMMR, 27(6): 1-8, 2018; Article no.JAMMR.43719
of 41% found by a mouthwash technique. That
study also depicted that C. albicans was not
uniformly distributed in the mouths of healthy
people. Also the risk factors for oral Candidal
infection in diabetic patients are complex. Some
authors emphasise the role of local factors such
as salivary flow rates and pH, poor glycemic
control and low salivary buffering capacity
[3,4,16].
C. albicans was the commonest yeast found on
subjects, followed by C. glabrata and C.
tropicalis. Same pattern was also observed by
Sanitá et al. [23] and Vanden Abbeele A et al.
[24].
5. CONCLUSION
Within the limitations of this in vivo study, the
following conclusions were drawn:
1. Candidal carriage and colonisation (based
on the CFU) is more in diabeticdenture
wearer patients than the non- diabetic
denture wearers.
2. In Group 1, shows positive growth on SDA
sample 1, sample 2 and sample 3
respectively.
In Group 2 (n=20), 0 (0%), 2 (10%) and 15
(75%) shows positive growth (p-
value1=0.001) on SDA sample 1, sample 2
and sample 3 respectively. Thus, Candidal
growth is dependent of time.
3. The common species of Candida
isolated in both diabetic and non-diabetic
denture wearer patients are: C. albicans,
C. glabrata and C. tropicalis.
CONSENT
Consent was taken individually from all patients.
ETHICAL APPROVAL
Ethical approval was taken from institutional
ethical research cell committee. Ethical approval
certificate number is CPGIDSH/562/17.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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_________________________________________________________________________________
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