International Journal of Dental

Research & Development (IJDRD)

ISSN(P): 2250-2386; ISSN(E): 2321-0117
Vol. 4, Issue 6, Dec 2014, 1-6
TJPRC Pvt. Ltd.

ISOLATION AND IDENTIFICATION OF CANDIDA SPECIES FROM VARIOUS

CLINICAL FORMS OF LEUKOPLAKIA AND ORAL LICHEN PLANUS
MERU S1 & NIVEDITA BAJANTRI2
1

Professor and Head, Department of Oral Medicine and Radiology, Uttaranchal Dental and Medical Research Institute
Mazrigrant, Dehradun, Uttarakhand, India

Professor, Department of Oral Medicine and Radiology, Teerthankar Mahaveer Dental College and Research Centre,
Moradabad, Moradabad, Uttar Pradesh, India

ABSTRACT
This study was carried out to isolate and identify the candida species in various clinical forms of leukoplakia and
oral lichen planus. Sixty patients with leukoplakia and 30 patients with oral lichen planus were selected. Yeasts were
isolated using swabs and inoculated in SDA medium. Specimen showing moderate to heavy growth was subjected to
speciation using the germ tube test, chlamydospore formation and sugar assimilation test. The results were statistically
evaluated for the test of significance using Chi square test and Fischers exact test.

KEYWORDS: Leukoplakia, Oral Lichen Planus, Candida

INTRODUCTION
Leukoplakia and lichen planus are the common precancerous lesions occurring in the oral cavity.1 The WHO
(1978) definition of leukoplakia was modified in 1984 as a whitish plaque that cannot be characterized clinically or
pathologically as any other disease and it is not associated with any physical or chemical causative agent except the use of
tobacco.2 A number of locally acting agents have been implicated in the etiology of leukoplakia. But tobacco consumption
still appears to be the most significant etiologic factor.3 In India; the etiologic influence of betel quid with tobacco chewing
is probably of much importance.4 it is common in the age group of 40-60 years. The male- female ratio as shown in recent
studies is 3:2.5
Candida species are indigenous opportunistic pathogens present as harmless commensals in the human oral cavity.
However, chronic oral candidiasis may be an etiological factor in oral leukoplakia, epithelial dysplasis and squamous cell
carcinoma. A comprehensive review of literature has concluded that there is considerable circumstantial evidence from
epidemiological, experimental and clinical studies that the candida species play a role in carcinogenesis. It has been shown
that a number of candida species are capable of endogenous nitrosamine production that result in carcinogenic N-nitroso
compounds, although this ability varies in different species.6
Lichen planus is a relatively common disease of unknown etiology involving skin and oral mucous membrane.
Oral lichen planus (OLP) affects from 0.1- 4% of population depending on the population sampled. It is a disease of the
middle aged to elderly people and the male female ratio is 1:2.7 The WHO classifies OLP into seven forms viz, reticular,
popular, plaque-like, erosive, ulcerative and bullous.

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Meru S & Nivedita Bajantri

Yeasts have also been identified in OLP lesions. There are no data indicating that yeast is involved in the etiology
of OLP, however, candida may be involved in the malignant transformation of OLP.8
The purpose of this study is to examine the prevalence and significance of candida infection in various clinical
forms of leukoplakia and oral lichen planus.

AIMS AND OBJECTIVES

To identify the various species of candida species seen in leukoplakia and oral lichen planus.

To find the association between various candida species with different forms of leukoplakia and oral lichen
planus.

To study the presence of candida species in different tobacco habits causing leukoplakia.

To compare the prevalence of candida species in leukoplakia with that of oral lichen planus.

MATERIALS AND METHODS

The patients for this study were selected from out patients who visited the department of Oral Medicine and
Radiology. Sixty patients with leukoplakia and thirty patients with oral lichen planus were selected. The diagnosis of
leukoplakia was made based on the WHO definition. All the selected patients except those with the typical clinical
presentation were subjected to biopsy. Patients with diabetes mellitus and patients on immune suppressant drugs were not
included in the study. Among the 38 male and 22 female patients selected with leukoplakia were in the age group of 36-55
and 16 females and 14 males with oral lichen planus were in the age group of 26-52.
Isolation of Yeasts
The samples were collected from the lesions using a sterile cotton swab. These swabs were subsequently
inoculated in Sabourauds dextrose agar (SDA) medium. The SDA is inoculated aerobically for 24-48 hours. Candida
grows as white, convex colonies. Samples growing 1-3 colony forming units were considered normal flora of the oral
cavity. The specimen showing moderate to heavy growth were subjected to tests for identification of species of candida.
Identification of Candida Species
The following tests were used to identify the candida species:

Germ Tube Test: The germ tube test is the standard test for identification of candida albicans. It involves
induction of hyphal growth (germ tube) from the yeast cultured in serum for 2-4 hours at 370C. Candida albicans
and Candida stellatoidea produce germ tube.

Chlamydospore Formation: Chlamydospore formation is induced by inoculation of the isolate on corn meal agar
plate. The inoculated area is overlaid with a coverslip and incubated at 220C for 72 hours. Candida albicans and
Candida stellatoidea are primarily associated with chlamydospore formation.

Sugar Assimilation Test: Sugar assimilation test was carried out using various sugar impregnated discs.
The discs were placed over the basal agar. The isolate was inoculated for 24-48 hours. Sugar assimilation profiles
for candida can be obtained by examining the zones of candidal growth around various sugar discs. The sugars
used were glucose, maltose, sucrose, lactose, trehalose and raffinose. (Table 1)

Impact Factor (JCC): 1.6658

Index Copernicus Value (ICV): 3.0

Isolation and Identification of Candida Species from Various Clinical Forms of Leukoplakia and Oral Lichen Planus

Table 1: Showing the Sugar Assimilation Properties of Various Candida Species

C. albicans
C tropicalis
C. stellatoidea
C. parapsillosis
C. krusie
C. guilimon
C. glabrata

Glucose
+
+
+
+
+
+
+

Maltose
+
+
+
+
+
-

Sucrose
+
+
+
+
-

Lactose
-

Trehalose
+
+
+
+
+
+

Raffinose
+
-

RESULTS
The study was conducted on 60 patients with leukoplakia in the age group of 36-55 years. The mean age was
found to be 45.5 years with 70% of patients in the age group of 36-45 years; 22.5 in 46- 55 years and 7.5% were 55 years
and above. There were 43 male and 17 female patients i.e. 71.6% male and 28.4% female patients. The sample consisted of
two types of leukoplakia- homogenous and speckled. Patient distribution among the two types were as shown in the
Table 2
Table 2
Type of Leukoplakia
Homogenous
Speckled

Number of Patients
52
8

Percentage
86.6
15.4

The various tobacco consuming habits were divided into-Chewing; Smoking; Both.The patient distribution among
various groups were as shown in the table 3
Table 3
Type of Habit
Chewing
Smoking
Both

Number of
Patients
30
18
12

Percentage
50
30
20

The study included 30 lichen planus patients with oral lesions in the age group of 26 to 52 years. This group
consisted of 16 males and 14 females. The sample consisted of patients with 2 clinical forms of oral lichen planus.
The patient distribution among the two clinical forms were as shown in the Table 4
Table 4
Type of Lichen Planus
Reticular
Atrophic

Number of Patients
21
9

Percentage
70%
30%

The study of prevalence of candida species in leukoplakia showed positive growth in 42 (70%) and negative in 18
(30%) of the patients. A test of significance Chi- square test resulted in a P value= 0.017 (p<0.05) indicating a significant
difference. The candida species isolated from various types of leukoplakia were as shown in the Table 5.
Table 5
Type of Leukoplakia
Homogenous
Speckled
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C. Albicans
31
8

C. Tropicalis
3
0
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The Fischers exact test resulted in P= 0.67 indicating the distribution of the two types f candida species in various
forms of leukoplakia do not differ significantly at 5% level of significance. The candida growth in leukoplakia patients
with various habits was as shown in Table 6.
Table 6
Candida Culture
Positive
Negative

Chewing
24
6

Smoking
9
9

Both
9
3

The P= 0.18 (p>0.05) therefore the proportion of patients with different tobacco habits showing the candida
growth does not vary significantly.
The study of prevalence of Candida species in oral lichen planus showed positive growth in 17 (56%) and
negative growth in 13 (44%) of patients. The test of significance using Chi- square test resulted in P value = 0.82
(p> 0.05). Therefore the prevalence of Candida species does not vary significantly in patients with oral lechen planus.
The Candida growth in various types of oral lichen planus was assessed and the results were as shown in Table 7
Table 7
Candida Culture
Type of lichen planus
Positive
Reticular
9
Atrophic
8

Negative
11
2

The Fischers exact test was carried out and the P value =0.119. The odds ratio for the prevalence of Candidal
growth among the two types of lichen planus was calculated and the value was = 6.66 indicating that it is likely to observe
candidal growth in atrophic type seven times more than in reticular type of oral lichen planus.
The distribution of candida growth among the symptomatic and asymptomatic patients was assessed and the result
was as shown in the Table 8.
Table 8
Candida Culture
Positive
Negative
9
3
8
10

Symptomatic
Asymptomatic

The Odds ratio =6, indicating that it is likely to observe Candidal growth 6 times more in symptomatic than in
asymptomatic patients.
The prevalence of Candidal growth in patients with leukoplakia and lichen planus were statistically evaluated and
results are as tabulated in Table 9.
Table 9
Candida Culture Leukoplakia Lichen Planus
Positive
42
17
Negative
28
13
The Odds ratio was found to be 1.9 (approx. 2). Therefore it is likely that the occurrence of Candidal growth in
Leukoplakia is twice as common as in oral Lichen planus.

Impact Factor (JCC): 1.6658

Index Copernicus Value (ICV): 3.0

Isolation and Identification of Candida Species from Various Clinical Forms of Leukoplakia and Oral Lichen Planus

DISCUSSIONS
Yeasts have been found to exist in the oral cavity in 2- 48% of individuals without any clinical symptoms.9
The role of Candida in production of the lesions is the essential problem. Three hypothesis may be suggested:

The presence of Candida can be regarded as coincidental i.e. a colonisation of an already diseased mucosa.

Candida may be responsible in part at least for the changes seen.

Candida may be the sole cause of the lesion.10

Mc Carthy and Shklar7 expressed that Candida grows readily on many pre-existing pathologic process.

Furthermore, Maiback H J and Kligman A M suggested that the defective epithelium may act as a predisposing factor for
the over growth of Candida albicans.11 Indeed it is well known that Candida species are keratophillic and they tend to
colonize in thick layers of keratin.12 Arendorf and Walker13 suggested that tobacco smoking might lead to localized
epithelial alteration which allows colonization by Candida. Candida Species have inducible enzyme system which allows
them to replicate using polycyclic aromatic hydrocarbons as their source carbon and energy.14 The present study showed
that 65% of the homogenous type of leukoplakia had positive growth while all of the patients with speckled leukoplakia
had positive Candidal growth. Roed- Peterson et al noted that Candida involved leukoplakias are often of the non
homogenous type.9 Whether the Candida are involved in the development of leukoplakia or just secondary invaders in
already established lesion is a matter to debate.
I M Lumdstrom15 noted that the incidence of positive culture in oral lichen planus was 44%. But this finding is
close to that in general population. The author also stated that the negative cultures obtained from the patients with oral
lichen planus may be due to the difficulty in obtaining a representative sample from a dry atrophic area.15 Earlier
investigations have demonstrated that the decreased salivary flow rate and salivary pH, raised blood and salivary glucose
levels may stimulate the growth of Candida albicans.15 Based on this phenomenon, in this study patients with diabetes
mellitus were excluded from the study sample. Hatchuel et al studied the prevalence of Candida in 185 biopsies of OLP
patients and candida infection was found in 34% of sample.16 there was no difference between ulcerative and non
ulcerative types. However Vincent et al showed that antifungal treatment resulted in marked symptomatic improvement in
89% and clinical improvement in 94% of patients.17
Roed- Peterson have found a relationship between the presence of Candida and occurrence of epithelial atypia.
However it is not clearly distinguished whether tissues with cellular atypia in leukoplakia are prone to invasion by the
organism or cellular atypia results from the invasion of Candida.11 The development of malignant condition in OLP lesions
has been reported.18 Krogh et al stated that yeasts may be involved in the malignant transformation in some cases of OLP.18
A number of Candida species are capable of producing carcinogenic N- nitroso compounds.6 These compounds
either directly act on the oral mucosa or interact with other chemical carcinogens to activate specified proto-oncogenes and
there by initiating oral neoplasia.9

CONCLUSIONS
This is a preliminary study to isolate and identify the Candida species associated with leukoplakia and oral lichen
planus. The study revealed a statistically significant prevalence of Candida species in leukoplkia. The Candidal invasion
was significantly more in speckled type of leukoplakia and atrophic type of oral lichen planus. A larger sample study is
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Meru S & Nivedita Bajantri

required to establish a relation between different species isolated in the oral lesions and their inherent ability to produce
carcinogens. This study gives scope to further studies with antifungal therapy in the management of leukoplakia and lichen
planus with a long term follow up in a larger sample group.

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Med Oral Pathol, 1987: 63: 48-54.

Impact Factor (JCC): 1.6658

Index Copernicus Value (ICV): 3.0