Relationships of Candida with Frequency of Brushing, Age and Smoking in Patients with Removable Dental Prosthesis: A Quantitative Study

Abstract

Objective: Although progress has been made to reduce opportunistic infection of fungi in the oral cavity, the prevalence of denture stomatitis associated with Candida has increased. The purpose of this study was to evaluate the effect of denture cleaning frequencies, age and smoking habit on the levels of Candida species in the saliva of patients wearing removable partial or complete dentures. Methods: Subjects wearing removable partial or complete dentures were recruited. A questioner was used to collect information on oral hygiene habits and smoking. Saliva samples were collected by oral rinse technique in a sterile container and cultured in duplicate Sabouraud Dextrose Agar. The numbers of colonies were determined by aCOLyte colony counter and the number expressed as a colony forming unit (CFU). The CFU and clinical data were analyzed for correlation and the Kruskal-Wallis test was used to determine statistically significant differences. Results: Among 99 subjects recruited, 47 were wearing complete dentures and 28 were smokers. The brushing frequencies were once (37 subjects), twice (39 subjects) and three times (23 subjects) per day. CFU was significantly higher in partial denture wearers than complete denture wearers and the brushing frequencies significantly correlated with CFU (r =-0.85, P= 0.001). There was no statistically significant difference in CFU between smokers and non-smokers and no correlation of CFU with age was found. Conclusions: This quantitative study has suggested that there are statistically significant differences in the levels of Candida in the saliva of subjects with different brushing frequencies and wearing a different type of denture. However, no statistically significant difference was noticed between smokers and non-smokers, and there was no significant correlation between CFU and age.

Full text

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Cite this article as: Ghalib AN, Gul SS, Abdulkareem JF. Relationships of Candida with Frequency of
Brushing, Age and Smoking in Patients with Removable Dental Prosthesis: A Quantitative Study. Sulaimani
Dent J. 2017;4(1):12-18.
Relationships of Candida with Frequency of
Brushing, Age and Smoking in Patients with
Removable Dental Prosthesis: A
Quantitative Study
Alan N. Ghalib 1, Sarhang S. Gul 2, Jwan F. Abdulkareem 3
Abstract
Objective: Although progress has been made to reduce opportunistic infection of fungi in the oral cavity, the prevalence of
denture stomatitis associated with Candida has increased. The purpose of this study was to evaluate the effect of denture
cleaning frequencies, age and smoking habit on the levels of Candida species in the saliva of patients wearing removable
partial or complete dentures.
Methods: Subjects wearing removable partial or complete dentures were recruited. A questioner was used to collect
information on oral hygiene habits and smoking. Saliva samples were collected by oral rinse technique in a sterile container
and cultured in duplicate Sabouraud Dextrose Agar. The numbers of colonies were determined by aCOLyte colony counter
and the number expressed as a colony forming unit (CFU). The CFU and clinical data were analyzed for correlation and the
Kruskal-Wallis test was used to determine statistically significant differences.
Results: Among 99 subjects recruited, 47 were wearing complete dentures and 28 were smokers. The brushing frequencies
were once (37 subjects), twice (39 subjects) and three times (23 subjects) per day. CFU was significantly higher in partial
denture wearers than complete denture wearers and the brushing frequencies significantly correlated with CFU (r = -0.85, P=
0.001). There was no statistically significant difference in CFU between smokers and non-smokers and no correlation of CFU
with age was found.
Conclusions: This quantitative study has suggested that there are statistically significant differences in the levels of Candida
in the saliva of subjects with different brushing frequencies and wearing a different type of denture. However, no statistically
significant difference was noticed between smokers and non-smokers, and there was no significant correlation between CFU
and age.
Keywords: Levels of Candida, Saliva, Denture wearer, Cleaning frequencies.
Submitted: June 14, 2017, Accepted: September 21, 2017
DOI: https://doi.org/10.17656/sdj.10066
1. Department of Microbiology, College of Medicine, University of Sulaimani, Sulaimani, Iraq.
2. Department of Periodontics, College of Dentistry, University of Sulaimani, Sulaimani, Iraq.
3. Department of Prosthodontics, College of Dentistry, University of Sulaimani, Sulaimani, Iraq.
* Corresponding author: alan.ghalib@univsul.edu.iq
Published by College of Dentistry, University of Sulaimani

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Introduction
Candida is a harmless commensal in the oral cavity
with a prevalence of 25-50% in healthy subjects(1)
and can cause disease when the relation between
host and microorganisms is disrupted(2). Several
local and systemic risk factors are related to
infection associated with Candida, such as
immunosuppression, antibiotics, age, smoking and
dentures(3).
Candida infections are very common amongst
denture wearers and this could be related to the
reason that wearing dental prosthesis is considered
as a predisposing factor for colonization of the
mouth with Candida species(4). A study by Pires et
al.(5) revealed that the prevalence of Candida ranged
from 60-100% in denture wearers. The rationale
behind the increased prevalence of Candida in
denture wearers could be that the transient
microenvironment underneath the denture favors
retention of food debris and consequently
microorganisms(5,6). Moreover, acrylic resin
dentures can act as a plaque retentive factor and
further enhance colonization of Candida(6).
Age is another factor linked to Candida infection,
since being elderly is usually associated with
systemic disease, decreased manual dexterity,
change in quantity and quality of saliva and thus
changes in the oral environment that enhance the
growth of Candida(7). Furthermore, Soysa and
Ellepola(8) demonstrated that cigarette smoking
increases overgrowth of Candida via its effect on
saliva and oral commensals, mainly Candida.
The increase in the number of Candida is the first
step toward the infection process and thus causing
different degrees of denture stomatitis of mucosa in
contact with the fitting surface of the denture(6,9).
Prevalence of Candida-associated denture stomatitis
has been reported in 60-65% of individuals with a
dental prosthesis(10), with clinical features of edema
and chronic erythema(8). Although denture
stomatitis mainly remains superficial and harmless,
it might cause bleeding of oral mucosa and coexist
with other conditions such as angular cheilitis and
burning sensation beneath the denture(11), taste
disorder(12) endodontic infection(13) as well as more
serious conditions such as oral cancer(14).
With population growth, in aging societies, there is
more likelihood of people losing their teeth and
having them replaced with a dental prosthesis(15).
Consequently, the chances of Candida-associated
denture stomatitis rise. Denture stomatitis caused by
a fungal infection in the human oral cavity(16) and
fungi can be detected on teeth surfaces, tongue,
cheeks, oral mucosa, dental prosthesis and
restorative material(17). However, rough surface
dental material harbors a higher number of
yeasts(18,19), which could be explained by the fact
that rough surfaces act as a retentive factor and
protect microorganisms from cleaning actions(20).
Several modalities have been proposed to provide
successful management of the patient with denture
stomatitis, such as correction of the nutritional
deficiency, advice on cessation of any smoking
habit, antifungal therapy and steroids. However,
appropriate oral hygiene practice remains the main
essential factor to remove Candida biofilms on host
tissues and dental prostheses(4).
This study is directed toward investigating the effect
of brushing frequencies, age and smoking habit on
the levels of Candida in the saliva of subjects with
removable partial or complete dentures.
Patients and methods
Study groups
Subjects wearing removable partial or complete
dentures were recruited for this study. The study
protocol was approved by the ethical committee of
University Sains Malaysia. Recruitment was
conducted among subjects attending the outpatient
clinic from January to March 2011 in the School of
Dental Sciences at University Sains Malaysia.
Potential subjects were screened by a clinical
investigator and evaluated for exclusion criteria
before being invited to take part in the study. The
exclusion criteria were patients with systemic
disease, active oral disease and history of antibiotic
in the last three months. After obtaining written
consent, the age, sex, frequency of brushing
(regardless of any method or materials used for
brushing), type of denture (denture’s age did not
take into account) and smoking habit were recorded,
followed by salivary sample collection.
Sample collection and Candida identification
Salivary samples were collected by oral rinse
technique. The participants instructed to rinse their
mouth thoroughly with sterile phosphate buffer
saline (10 ml) for 60s and the sample collected in a
sterile container, stored in a cool box and then
transported to the oral microbiology laboratory for
processing. The oral rinse sample centrifuged at

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1700g for 10 min then the supernatant discarded.
One ml of concentrated saliva sample used for
culturing. Serial decimal dilutions of samples were
prepared in Phosphate-buffered saline. Aliquots of
0.1 ml were cultured in duplicate on Sabouraud
Dextrose Agar (Himedia, India) and incubated at
370C for 48 hrs(21).
The growth of Candida species was identified by
smooth, white or creamy colored buttery colonies
(Figure 1). Candida species were further identified
by Gram stain as Gram-positive budding shaped
yeast cells (Figure 2). The numbers of colonies were
determined using the aCOLyte colony counter
(Symbiosis, USA) (Figure 3 and 4). A pilot study on
five samples was conducted to choose the
appropriate dilution. The samples above or below
the sensitivity (30- 300 CFU) of the machine were
reanalyzed and the final numbers of CFU per mL
(mean of duplicate samples) were obtained taking
the dilution factor into account.
Statistical analysis
The normality test (Shapiro-Wilk test) for
continuous data was performed and the data were
then subjected to an appropriate test. The
statistically significant difference between cleaning
frequencies was conducted by Kruskal–Wallis test.
The Mann-Whitney test was used to determine
statistically significant differences in CFU between
partial and complete denture wearers and smoker
versus non-smoker. Spearman’s correlation was
used to determine the correlations of CFU with age
and brushing frequencies. Statistical significance
was defined as p ≤ 0.05 and all calculations were
conducted using the SPSS software package
(version 21; SPSS Inc., Chicago, IL, USA). The null
hypothesis was that none of the above variables
associated with CFU. Advice on data analysis was
provided by an expert statistician.
Results
Ninety-nine subjects (38 male and 61 female) were
recruited with the mean age of 59.9 ± 10, ranging
from 39 to 80 years (18%: 39-49 years, 32%: 50-59
years, 32%: 60-69 years, 18%: 70-80 years). Forty-
five subjects were wearing complete dentures and 28
subjects were smokers. Thirty-seven subjects
cleaned their denture once a day, 39 subjects twice a
day and 23 subjects three times a day.
Candida species were isolated from 99% of the
subjects. Analysis of saliva samples showed a
statistically significant difference in CFU levels in
partial denture wearers (median 1.5 106 mL-1)
compared to complete denture wearers (median 1.1
106 mL-1) (Mann-Whitney test, P = 0.01) (Figure 5).
Moreover, the Kruskal-Wallis test demonstrated
statistically significant differences (P <.001) in CFU
levels between subjects cleaning their denture once
(median 2.3 106 mL-1), twice (median 1.1 106 mL-
1) and three times a day (median 0.5 106 mL-1)
(Figure 6). There was no statistically significant
difference in CFU levels between smoker (median
1.2 106 mL-1) and non-smokers (median 1.3 106
mL-1) individuals (Mann-Whitney test P> 0.05)
(Figure 7).
Spearman’s correlation also showed a statistically
significant correlation between CFU levels and
cleaning frequencies (r= 0.8, P<.001). There was no
statistically significant correlation between CFU
and age (P= >0.05).
Figure 2: Gram stain showing Candida as gram-
positive round cells.
Figure 1: Candida growth on Sabouraud Dextrose
Aga
r
.

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Figure 4: aCOLyte colony counter connected to the
computer.
Figure 5: Comparison of median CFU between partial and complete
denture wearers.
Discussion
This study found that the number of Candida species
decreases significantly with increased cleaning
frequencies and is statistically significantly higher in
partial denture wearers than complete denture
wearers. However, there were no statistically
significant differences between smokers and non-
smokers plus no correlation between CFU and age
of subjects was detected. To our knowledge, this is
the first time that the number of Candida species in
the saliva of removable denture wearers has been
investigated against frequencies of cleaning the
denture. As denture brushing frequencies increased,
the numbers of Candida in the saliva decreased
(Figure 6). Furthermore, cleaning frequencies were
inversely proportional to the levels of Candida
species in saliva (r= 0.8, P= <.001). Moreover,
levels of Candida were demonstrated to be
statistically significantly higher in partial denture
wearers than complete denture wearers, which might
be associated with the presence of teeth in partial
denture wearers that provide further surfaces within
the oral cavity that are difficult for patients to clean,
a result that is in accordance with data published by
Gusmão et al.(22).
Figure 3: aCOLyte colony counter from symbiosis.

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Figure 7: Comparison of median CFU between smoker and non-smoker subjects.
There are conflicting data regarding the association
between the number of Candida species in oral
cavity and smoking habit. Studies by Abu-Elteen
and Abu-Alteen(23) and Shin et al.(24) demonstrated
higher levels of Candida species in oral cavity of
smokers than that of non-smokers, which is not in
agreement with the result of this study and this could
be due to the fact that in this study the number of
cigarettes per day and duration of smoking was not
taken into account. However, our data were in
accordance with other studies conducted by Masipa
et al.(25) and Darwazeh et al.(26).
Figure 6: Comparison of median CFU in subjects with different denture
brushing frequencies.

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Again there are conflicting results regarding the
relationship between a number of Candida and age
of subjects. The data of this study did not show any
correlation between age of patients and number of
CFU, and this is not in line with Loster et al.(27) that
showed a higher prevalence of Candida in complete
denture wearers above 50 years old. The related
points to consider are in this study both complete
and partial denture wearers included and our sample
size is not as large as of Loster et al.(27). Furthermore,
82% of subjects investigated in this study were
above 50 years old. Consequently, there were not
enough samples below 50 years old to compare.
Conclusions
This study has demonstrated that subjects with
higher cleaning frequencies have lower levels of
Candida species and are thus less likely to develop
denture stomatitis. Partial denture wearers need to
clean their denture and remaining teeth more
frequently, as higher levels of Candida were
detected. Further studies on duration of cleaning,
different cleaning techniques and cleaning materials
need to be conducted to reveal the exact effect of
brushing on the number of Candida. Furthermore, a
longitudinal study needs to be carried out to show
the recolonization time after denture brushing and
the effect of brushing frequency on the reduction of
denture stomatitis.
]
Acknowledgments
The authors would like to thank the ethical
committee at University Sains Malaysia (USM) and
the School of Dental Sciences at USM for their
support.
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