International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391
Assessment of Oral Sample Collection Technique
for the Isolation of Candida albicans from Patients
having Denture Stomatitis
Hadeel Salman Alazzawi1, Jamal Noori Ahmed2
1
Ninavah Health Directorate, Ministry of Health, Iraq
2
Department of Oral Diagnosis, College of Dentistry, Baghdad University, Iraq
Abstract: Candida associated denture stomatitis remains the more frequent form of oral candidiasis and specifically seen among
denture wearers. Denture stomatitis is inflammatory lesion of the denture bearing area caused by Candida albicans. Direct laboratory
methods such as microscopy provide preliminary information about the microbial involvement in an infection, but microbial growth is
usually required for definitive identification and characterization. There are number of methods used in the sample collection. This
investigation has assessed the techniques used in the collection of the fungal specimen. Thirty seven patients who had Candida induced
denture stomatitis were selected clinically according to Newton`s criteria. For each patient, three methods of sample collection were
done to investigate the presence of Candida colony. The methods were colony forming unit, swab from palatal mucosa, swab from saliva
and salivary culture. The samples were cultured in Sabouraud dextrose agar for 48 hours, then semiquantitive and quantitive measure
of the colonies were assessed. The results were high significant relation between the three methods. So that, any of the three methods of
oral sample collection can be used to determine oral Candida count.
Keywords: oral, Candida, microbiology
1. Introduction diagnosis and a decision on antimicrobial therapy. Hence,
dentists should be acquainted with the techniques of taking
Candida associated denture stomatitis is a common specimens for laboratory analysis [6], [9]-[11].
inflammatory process affecting about 60% of the subjects
carrier of a prosthesis [1], with preferential localization to A standard technique has been followed. Solid media are a
the palatal mucosa. Among the predisposing local factors the useful media as it facilitate observation of colonial
main one is the accumulation of microbial plaque especially characteristics and helpful in identification of organisms, it
Candida albicans which has the highest adhesion capacity to also facilitate quantification of organisms as in colony
oral epithelial cells and on the surface of the denture in forming unit (CFU) [6], [ 9], [ 11].
contact with the mucosa [2], [3].
In culture, a scrape can be cultivated on Sabouraud Dextrose
The clinical diagnosis of any of the forms of oral candidiasis agar (SDA) which contains glucose and modified peptone
is based on recognition of the lesions, which can be (pH 7.0), then incubated for 48-72 hours. When Candida
confirmed by the microscopic identification of Candida in albicans appears as cream-coloured large convex colonies,
the oral samples and/or isolation in culture, among other the result is expressed as colony forming units per milliliter
diagnostic methods [4]-[7]. Although the clinical diagnosis (CFU/mL). This method is a valuable adjunct in the
of oral candidiasis is relatively simple, confirmation of the diagnosis of erythematous candidiasis and denture stomatitis
diagnosis can be made by the microscopic morphological as these infections consist of homogeneous erythematous
criteria observation of Candida in oral lesion samples which lesions [12]-[14].
can be made with fresh samples using special stains and by
isolation in culture (macroscopic morphological criteria) [7], Salivary culture techniques are used in parallel with other
[8]. diagnostic methods to get an adequate quantification of
Candida colonies [15]-[18]. Saliva is collected and number
Specimen collection is the process taking microbes from the of candidal colonies counted after culture on Sabouraud agar
infection site (in vivo) by some means of collection. After [19].
that, growing them in the artificial environment of the
laboratory (in vitro). Once grown in culture, most microbial 2. Patients, Materials and Methods
populations are easily observed without microscopy and are
present in sufficient quantities to allow laboratory The sample of the study were 37 patients recruited from
identification procedures to be performed [9]. The objective Baghdad College of Dentistry clinics and from specialist
of inoculating the specimen or a culture of fungus on to a dental health centers in Baghdad city. Patients fulfilled the
solid medium is to obtain discrete colonies of organisms clinical selection criteria of oral candidiasis with C. albicans
after appropriate incubation. Diagnostic microbiology positive culture have entered the clinical trial. Approval was
involves the study of specimens taken from patients obtained from the local authorities to conduct the trials.
suspected of having infections. The end result is a report that Clinical signs consistent with oral candidiasis in palatal
should assist the dentist or clinician in reaching a definitive
Volume 6 Issue 4, April 2017
www.ijsr.net
Licensed Under Creative Commons Attribution CC BY
Paper ID: ART20172380 DOI: 10.21275/ART20172380 1100
International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391
mucosa were categorized according to Newton`s criteria less than the 0.05 level of significance was considered
[20] as follows: statistically significant.
0: no inflammation; Grade 1: pinpoint hyperaemia; Grade 2:
generalized erythematous type and Grade 3: hyperplastic
granular type.
Mucosa swab from palate of the patients was taken. Mucosal
oral samples were obtained by a sterile cotton tip applicator
(figure 1), swab was collected from the palatal mucosa by
weeping all palatal area then plated immediately in SDA
then sent to microbiology laboratory for incubation and
isolation of Candida.
Unstimulated Saliva Collection was made when subjects
were instructed to remove their dentures, and whole
unstimulated 2 ml of saliva was allowed to pool in a
disposable sterilize plastic container. Other swab was taken
from saliva and also cultured. Figure 1: Swab
By micropipette, 10 μml of the sample was plated 3. Results
immediately to the SDA plate for colony forming unit
(CFU) count. Total of 37 patients have participated in this study, (12
males, 25 females), their age ranges between 30-75 years.
For culturing and isolation of Candida albicans, swab of The mean age ± SD was 55.7± 12.24.
mucosa and saliva sample were streaked onto SDA
(containing 40 mg/ml of Chloramphenicol) plates, Clinically, pin point erythema were 9 cases, diffuse
semiquantitively struck out in four quadrants, the swab was erythema were 17 while papillary hyperplasia were 11 cases.
applied by a dime-sized area. The inoculum is then crossed- Microbiologically, in macroscopic examination, colony
struck with a loop. The loop is then flipped over or flamed characteristics were observed after 48 hours as creamy color
and quadrant two was struck by pulling the loop through with soft consistency and yeasty odor on media. The surface
quadrant. The loop is then flamed again and quadrant three and margins of the colonies were smooth.
was streaked by going into quadrant two and streaking the
rest of the area. Quadrant four was streaked by pulling the Counting of the colonies in mucosal (figure 2) and saliva
loop over the rest of the agar without further flaming. After plates was done for convenient presentation of the data,
that incubated aerobically at 37 °C for 48 hours. CFU/ml was adopted to plot the findings (figure 3).
For CFU, salivary fluid streak line was crossed-struck with a The three different methods of microbiological
loop to produce isolated, countable colonies [9], [12]. measurements were correlated with each other, table 1
showed the difference in median of microbiological
The samples were identified using first culture measurements mucosa and saliva swabs with CFU ordered
characteristics by detecting the possibility of Candida categories with very strong correlation between salivary
albicans being present in the SDA, by describing shape, swab counting and salivary CFU methods (P< 0.001). Table
color and size of the yeast colonies grown on agar. 2 showed the difference in median mucosal colony count
with saliva colony count in swab methods, the statistical
Gram stain and germ tube was done to examine results showed high significant relation between the two
microscopically. Candida count was done for each culture methods.
after 48 h of incubation period.
Statistical analysis: Data were translated into a computerized
database structure. An expert statistical advice was sought
for. Statistical analysis was computer assisted using SPSS
version 21 (Statistical Package for Social Sciences).
The outcome quantitative variables in the current study were
non-normally distributed. Such variables are described by
median and interquartile range. The difference in median of
a quantitative non-normally distributed variable between 3
groups was assessed by the non-parametric test Kruskal-
Wallis.
The statistical significance, direction and strength of linear
correlation between 2 quantitative variables were measured Figure 2: Candida albicans from palatal mucosa sample on
by Spearman’s rank linear correlation coefficient. P value SDA culture.
Volume 6 Issue 4, April 2017
www.ijsr.net
Licensed Under Creative Commons Attribution CC BY
Paper ID: ART20172380 DOI: 10.21275/ART20172380 1101
International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391
4. Discussion
The diagnosis of an infectious disease entails a number of
decisions and actions by many people. The clinician or
dentist collect an oral microbiological sample, send to
laboratory and then the dentist receives the laboratory report
and uses the information to manage the condition [6], [8].
The present study offers three methods of the sample
collection of oral candidiasis, with the purpose of
establishing when complementary microbiological
techniques for the investigation of oral candidiasis should be
used, and which techniques are most commonly employed in
routine clinical practice in order to establish a definitive
diagnosis.
Figure 3: Candida albicans CFU from salivary sample on
SDA culture.
The three different methods have been used in collecting
microbiological sample. Swab from palatal mucosa; swab
Table 1: The difference in median of selected
from saliva and salivary CFU) is easy and acceptable by the
microbiological measurements (mucosa and salivary swabs)
patient. The scraped methods are successfully used in
with CFU.
detecting Candida [12], [13], and considered as a simplified
CFU /ml quartiles
Fourth
diagnostic aid [4]. However, Terai and Shimahara, 2009
(highest) concluded that swab and culture test, which are currently
First (lowest) Average quartile used in the diagnosis of oral candidiasis, can yield false-
quartile (<= 2) (3 - 125) (126+) P negative results in 25% of cases [21].
Mucosal colony
count 0.032 In case of salivary method, many studies also confirm that
(0 to salivary candidal shed can be used as a tool in predicting
Range (0 to 13) 151) (0 to 283) oral candidiasis [15], [16], [18]. Malamud and Rodriguez-
Median 2 6 25 Chavez in 2011 recommended that uses of saliva samples
Interquartile for Candida pathogen isolation for clinical diagnosis of
range (0 to 5) (1 to 8) (6 to 37)
fungal infections [17].
N 10 18 9
Mean rank 12.2 19.8 24.9
r=0.54 P<0.001
The higher significancy between three techniques indicated
Saliva colony that all of these techniques can be used in determining
count <0.001 Candida count. However, this research could not determine
(1 to the superior method. Therefore additional study is needed to
Range (0 to 2) 120) (11 to 150) investigate the relation between the clinical criteria and
Median 0 10 93 candidal count.
Interquartile
range (0 to 0) (4 to 35) (80 to 137) 5. Conclusion
N 10 18 9
Mean rank 5.6 20.5 30.9
The three different methods of collecting microbiological
r=0.938 P<0.001
samples (swab from palatal mucosa; swab from saliva and
salivary CFU) can be used in determining Candida count.
Table 2: The difference in median mucosal colony count
with saliva colony count 6. Acknowledgement
Saliva colony count quartiles
Fourth The microbiological work was done at Microbiology Unit in
First (lowest ) (highest)
Laboratory Teaching Department (Directorate of Medical
quartile Average quartile
(<= 1) (2 - 67) (68+) P City) in Baghdad. We acknowledge the help of Mrs. Bassma
Mucosal colony AL-Mosleh at Microbiology unit. Of course, none of this
count 0.009 would have been possible without her advice.
(0 to
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Paper ID: ART20172380 DOI: 10.21275/ART20172380 1102
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