Prevalence of oral parameters in smokeless tobacco-associated precancer.

AIM: To evaluate and correlate the prevalence of Candida species, salivary flow rate, pH, and buffer capacity of oral cavity in subjects with leukoplakia, tobacco pouch keratosis, and healthy controls.
MATERIALS AND METHODS: In this prospective study, a total of 25 cases of leukoplakia, 25 of tobacco pouch keratosis, and 50 of healthy controls were studied. Sabouraud's dextrose agar (SDA) for the identification of Candida and HiCrome Candida differential Agar for the species identification of Candida were used. Calculation of salivary flow rate, pH, and buffer capacity was done with Saliva-Check Buffer kit (GC).
RESULTS: The prevalence of Candida carriage obtained was considerably greater in the study group associated with smokeless tobacco use. NonCandida albicans Candida species predominated over Candida albicans in speciation among study groups on HiCrome agar and Candida albicans predominated among control groups. Statistically significant values were obtained by comparing the salivary flow rate, pH, and buffer capacity between the study groups and control groups.
CONCLUSION: The present study indicated Candida prevalence was seen more in study group than healthy control groups, suggesting increased Candida in smokeless tobacco users. The prevalence of NonCandida albicans Candida species among leukoplakia and tobacco pouch keratosis indicates that there is pervasiveness of significant NonCandida albican Candida species in relation to salivary flow rate and buffer capacity. Therefore, the choice of antifungal treatment promptly must deal with not only resistance but, similarly, with the transformation in predominance from Candida albicans to NonCandida albicans Candida species. The numbers of sample study were considerably less to conclude such result hence, numbers of studies should be performed at larger scale in the particular area. Copyright:

Introduction

The effect of smokeless tobacco has always been ignored in context to potentially malignant disorders in comparison with smoking tobacco.[1] However, researchers have acknowledged smokeless tobacco use as a leading cause of cancer[2] and also smokeless tobacco leads to leukoplakia, tobacco pouch keratosis, oral submucous fibrosis (osmf), periodontal infection, delayed wound healing, and dental caries.[3] Smokeless tobacco comes in two principal forms i.e., snuff and chewing tobacco. The quid stick in the oral cavity for hours and the saliva gets mixed with tobacco juice. This way chemical carcinogens in smokeless tobacco are released in the oral cavity i.e., polynuclear aromatic hydrocarbons such as benzo[a] pyrene, polonium 210, N-nitrosamines, and other chemicals include radium 266 and lead 210 which form adducts and cause DNA damage and ultimately leads to malignant transformation.[4] Investigations in experimental animals revealed that benzo[a]pyrene and one of its metabolites leads to oral and perioral skin cancer.[5] Nicotine, one of the principal constituent, exerts a negative reaction at the cellular level by altering metabolism and invades the contacts between tissues which advances cancer, altering cellular changes. It also affects microflora of the oral cavity, by increasing the number of destructive bacteria and pathogenic fungi.[6] It mixes with plasma proteins, and attaches to the mucous membrane depending on pH, higher the pH greater the lipophilicity; vigorous resorption by an organic membrane which further causes xerostomia and supports the colonization of Candida. Nicotine also decreases salivary pH by the accumulation of calcium and phosphorus ions, and its addiction is a critical factor in the progress of potentially malignant disorders such as leukoplakia that increases the adherence of Candida fungi in the oral cavity, making lesion more vulnerable to malignant transformation.

Smokeless tobacco consumption initiates oral cancer[5] hence it is necessary for the dental professional to identify the possibility and duration of tobacco-associated leukoplakia and tobacco pouch keratosis transformation into malignancy. Tobacco pouch keratosis is a white lesion at the site which comes in contact with tobacco continuously [Figure 1], the lesion appears thickened, leathery, and oral leukoplakia [Figure 2].[7] There is much debate about whether Candida species should be considered in the etiology or progression of oral leukoplakia and tobacco pouch keratosis, and many investigators have identified the manifestation of Candida albicans frequently with nonhomogeneous type leukoplakia.[8]

Figure 1

Intraoral photographs showing tobacco pouch keratosis (Tobacco User > 10 years)

Figure 2

Intraoral photograph of the patients showing a white patch in the oral cavity of patients diagnosed as Leukoplakia (Tobacco user < 5 years)

We designed the prospective study including the pervasiveness of Candida species and different nonCandida albicans candida species identification, assessment of salivary flow rate, pH, and buffer capacity associated with smokeless tobacco among subjects with leukoplakia and tobacco pouch keratosis in comparison with healthy control subjects; since fewer studies in the record are available regarding this in comparison to potentially malignant disorders associated with smoking tobacco. This study could be a possible indicator for the awareness of potential complications and health hazards in the individual having the habit of smokeless tobacco.

Materials and Methods

The present study was conducted in the Department of Oral Pathology, Microbiology, and Forensic Odontology HIDS Paonta Sahib, India and a total of 100 subjects were included. Out of which 25 were clinically diagnosed with leukoplakia (20 nonhomogeneous, 5 homogenous), 25 were diagnosed with tobacco pouch keratosis and 50 were healthy controls.

Leukoplakia:

The subjects were categorized according to WHO 1980 classification

Homogenous leukoplakia: uniformly white and unscrapable.

Nonhomogeneous leukoplakia: white and speckled with red surface

Tobacco pouch keratosis:

White patch located at the point where the tobacco is placed in the mouth.

Inclusive criteria

Study group:

The cases nonsmoking >20 years or never smoked

Chewing tobacco for at least 5 years.

Not taking any medicine (antibiotic/fungal/corticosteroid) for 6 months.

With no systemic conditions.

Not wearing any kind of prosthesis.

Absence of decayed teeth

Healthy control:

No history of taking any form of tobacco such as smokeless/smoking, not taking any medicine (antibiotic/fungal/corticosteroid) for 6 months.

With no systemic conditions.

Not wearing any kind of prosthesis.

Absence of decayed teeth

Sabouraud dextrose agar (SDA) was used to identify Candida among the groups and then the HiCrome Candida differential agar media was used to further evaluate various strains of Candida species among the subjects. The salivary samples were collected between 9–11.30 a.m. in both the study and control groups. The salivary flow rate, pH, and buffering capacity were measured using the “Saliva-check BUFFER kit [Figure 3]” of GC Corporation. The kit is provided with pH strips, which measures the pH from 5–8, saliva collection cups, saliva dispensing pipette, and buffer test strips.

Figure 3

(a) Saliva dispensing cup. (b) Inv itro Buffer strip. (c) Invitro pH strip

Salivary flow rate

A healthy adult produces about 500–1500 ml of saliva per day with an average rate of about 0.5 ml/min–1.6 ml/min. In the present study, the unstimulated salivary flow was checked. Patients were instructed to expectorate into the spittoon. The salivary sample was collected into the collection cup at regular intervals. The quantity of saliva was measured by checking the ml markings on the side of the cup [Figure 3a].

Quantity of saliva in 5 mins

Measured according to the reading:

< 3.5 ml: very low

Between 5.0–3.5 ml: low

> 5.0 ml: normal

Buffering capacity

A buffer is a solution that tends to maintain a constant pH. If the pH starts falling after the ingestion of a substrate then it returns to the original resting level after a period of time due to inherent buffers present in the saliva.

Buffer test strip from the foil package was taken out and placed on an absorbent tissue with the test side up. Using pipette, sufficient saliva was withdrawn from the collection cup and dispensed one drop on each of the three test pads. The strip was immediately turned up to 90° to soak up excess saliva on the absorbent tissue. The test pad began to change color immediately and after 2 mins the final results were calculated by adding the points according to the final color of each pad [Figure 3b].

Readings were measured according to:

Green: 4 points

Green/blue: 3 points

Blue: 2 points

Red/blue: 1 point

Red: 0 points.

Results were interpreted in the combined total.

0–5: very low

6–9: low

10–12: normal/high.

pH

The value of critical pH is usually about 5.5 ranging anywhere between 5.2 and 5.7. For the pH evaluation, the patient was instructed to expectorate pooled saliva into the collection cup, the pH strip was placed in the sample for 10 s and then color change on the strip was checked and compared with the testing chart available in the package [Figure 3c].

Readings were interpreted according to the following color change:

Highly acidic saliva: red section, pH 5.0–5.8.

Moderately acidic saliva: yellow section, pH 6.0–6.6.

Healthy saliva: green section pH 6.8–7.8.

Colony forming units (CFU)

For CFU, the samples were taken by oral-rinse technique, subjects were asked to rinse with 10 ml saline and expectorate, then 100 μL was taken and samples were inoculated on the plate and incubated for 48 h at 37°C in the incubator, and later colonies were calculated under the colony counter and speciation of Candida was done on the basis of distinctive morphological characteristics [Figure 4].

Figure 4

Candida colonies on HiCrome Candida differential agar: Candida krusie (pink colored), Candida albicans (green colored), Candida glabrata (cream colored), Candida tropicalis (purple colored)

Result

Total subjects included in the present study were in the age range from 20–70 years. Among 25 total subjects of leukoplakia, 20 subjects were the nonhomogeneous type with the patch on buccal mucosa in 12 subjects and 8 in the vestibule while 5 subjects were with homogenous type leukoplakia. Out of total 25 subjects with tobacco pouch keratosis 19 were with pouch in the posterior vestibular part of oral cavity and 6 subjects with tobacco pouch in the anterior vestibule in Table 1.

Table 1

Total number of subjects included in the study

Subjects	Leukoplakia		Tobacco pouch keratosis	Healthy controls
	Homogenous	Nonhomogeneous
100	5	20	25	50

To analyze the prevalence of Candida species among study and control groups we applied the Cross Tab test. Candida species on the HiCrom Candida differential agar media were differentiated on the basis of color and morphology [Figure 4] in Table 2. In study groups we found different species in the following descending order:

Table 2

Prevalence of Candida species obtained among groups on HiCrome agar was identified on the basis of color and morphology and following percentages were obtained by applying Cross tab test

Candida species identified in %	Study group			Control group
	Leukoplakia	Tobacco pouch keratosis	Total
C. albican	20.0%	31.0%	51%	31.9%
C. glabrata	31.1%	26.2%	57.3%	23.6%
C. krusie	4.4%	0.0%	4.4%	2.8%
C. tropicalis	44.4%	42.9%	87.3%	41.7%

Leukoplakia:

C. tropicalis > C. glabrata > C. albicans > C. krusie

Tobacco pouch keratosis:

C. trpoicalis > C. albicans > C. glabrata

Healthy Controls:

C. tropicalis > C. albicans > C. glabrata > C. krusei

Data indicates that numerous Candida species are present in study groups than control groups. In addition, increased non Candida albicans Candida species were obtained among all the groups.

For the statistical analysis of CFU among the study group and control group, we applied Annova test and significant value for CFU i.e. P ≤ 0.001 were obtained as tabulated in Table 3. Indicating that in the subjects with leukoplakia and tobacco pouch keratosis associated with smokeless tobacco, Candida carriage rate was found to be significantly increased and this can be related to smokeless tobacco consumption.

Table 3

Statistical significant prevalence of CFU among study groups and the control group was obtained by applying Annova test

Subjects	CFU/ml
Study group
Leukoplakia	P≤0.001
Tobacco pouch keratosis	P≤0.001
Control group	P≤0.001

For analysis of the statistical value for salivary flow rate, we applied post-hoc test and statistically significant value for salivary flow rate were P ≤ 0.001 among leukoplakia and healthy controls and P ≤ 0.001 among tobacco pouch keratosis and healthy controls obtained in Table 4. A significant decrease in salivary flow rate among study group in comparison with control group indicates smokeless tobacco consumption.

Table 4

Statistical analysis for salivary flow rate among the study group and control group was obtained by applying post-hoc test

Subjects	Number	Mean	Minimum/5 min	Maximum/5 min	P
Study group
Leukoplakia	25	3.14	2	5	P≤0.001
Tobacco pouch keratosis	25	2.66	2	5	P≤0.001
Control group	50	4.022	2	6	P≤0.001

Similarly for the analysis of pH among study group and healthy controls, we applied post-hoc test and statistically significant values for pH were obtained i.e., P ≤ 0.001 among leukoplakia and healthy controls and P ≤ 0.001 among tobacco pouch keratosis and healthy controls in Table 5. The data indicates that more acidic pH is obtained among the study group and significant value was obtained thus, it can be inferred that decrease in pH is related to smokeless tobacco consumption.

Table 5

Statistical analysis for pH among the study group and control group was obtained by applying post-hoc test

Subjects	Number	Mean	Minimum	Maximum	P
Study group
Leukoplakia	25	5.93	3.2	6.8	P ≤ 0.001
Tobacco pouch keratosis	25	6.21	4.4	7.2	P ≤ 0.001
Control group	50	7.02	6.4	7.8	P ≤ 0.001

For the statistical analysis of buffer capacity, we applied post-hoc test and statistically significant value for buffer capacity was obtained as P ≤ 0.001 among leukoplakia and healthy controls and P ≤ 0.001 among tobacco pouch keratosis and healthy control in Table 6. The obtained data indicate that significantly decrease in buffer capacity among study groups can be related to smokeless tobacco consumption.

Table 6

Statistical analysis for buffer capacity among the study group and control group was obtained by applying post-hoc test

Subjects	Number	Mean	Minimum	Maximum	P
Study group
Leukoplakia	25	2.12	1	3	P≤0.001
Tobacco pouch keratosis	25	2.40	1	6	P≤0.001
Control group	50	5.94	1	12	P≤0.001

Statistical correlations of CFU with salivary flow rate, pH, and buffer capacity were obtained by applying Pearson's test ad on comparing CFU among each group with salivary flow rate, pH, and buffer capacity, statistically significant value was obtained as P ≤ 0.01 in leukoplakia group and P ≤ 0.03 in tobacco pouch keratosis group 0.03 while on comparing CFU with salivary flow rate and buffer capacity with pH a statistically significant value of P ≤ 0.01. Among healthy controls no significant relationship was obtained. The data obtained are tabulated in Table 7. The data indicate that Candida carriage rate in the oral cavity is influenced by the salivary flow rate, pH, and buffer capacity. The statistically significant value obtained among study groups can be related to smokeless tobacco consumption.

Table 7

Statistical the correlation between CFU and salivary Flow rate, CFU and pH, and CFU and buffer capacity, Pearson test was used and the following results were obtained

Group	CFU and Salivary flow rate		CFU and pH		CFU and Buffer capacity
	r	P	r	P	r	P
Study group
Leukoplakia	−0.58	<0.01*	−0.63	0.01*	−0.56	<0.01*
Tobacco pouch keratosis	0.39	0.03*	−0.46	0.01	0.42	0.03*
Control group	−0.08	0.91	−0.11	0.87	−0.04	0.92

*Statistically significant

Discussion

It has already been established that long-term smokeless tobacco consumption leads to potentially malignant disorders. Clinical significance of the present study can be explained on the basis that smokeless tobacco has higher level of carcinogen present and leads to potentially malignant disorders which further leads to the malignant transformation eventually. Candida infected leukoplakia makes lesion more vulnerable for malignant transformation and there are few studies present in literature on potentially malignant disorders associated with smokeless tobacco consumption and their relation with Candida prevalence, salivary flow rate, pH, and buffer capacity. In the present study, we have obtained statistically significant results for the presence of Candida among study groups, who were associated with smokeless tobacco use (leukoplakia and tobacco pouch keratosis) on comparing with healthy controls, who were not associated with use of smokeless tobacco. The obtained results can be explained on the basis as smokeless tobacco releases nitrosamines in the oral cavity which changes the oral cavity microflora, increasing the number of harmful bacteria and pathogenic fungi. Longer duration use of smokeless tobacco leads to premalignant disorders e.g. leukoplakia, pathologic variations in the mucous membrane or tongue, and tumor. It also decreases salivary pH and increases phosphorus-ion concentration resulting in a significant acidic environment and leading to dryness of the mouth, xerostomia, all the above-mentioned factors increase the accumulation of Candida in the oral cavity,[6] otherwise the subjects included in the study group were healthy.

There has always been a debate about whether Candida is the cause or a superimposed infection in a lesion. In our study, study groups were associated with smokeless tobacco consumption and smokeless tobacco contains high level of carcinogen nitrosamines, which binds to DNA to form adducts causing miscoding or irregularities in DNA replication. Thus results in oncogene formation and initiates cancer formation. On the basis of above findings, Candida in the present study can be concluded as a superimposed infection in a lesion, which makes the lesion more vulnerable to malignancy by showing phenotypic change from yeast to hyphae form and producing carcinogen like nitrosamines. This leads to initiation of cancer development.[7] There are studies in literature both contradictory and similar in nature to the result found in our study e.g., Peterson et al. and Daftary et al.[9] concluded etiological role of Candida infection to be more significant. Bancozy (1977)[10] observed statistically significant results that Candida albicans infections act as an etiological factor in malignant conversion of leukoplakia. Krogh et al.[11] showed significant malignant potentials of Candida in nonhomogeneous leukoplakia than homogenous forms.

Another finding of the study is the prevalence of non Candida albicans Candida species among study groups mainly C. tropicalis. Studies in favor e.g. Connolly et al.[12] explained similar results among smokeless tobacco users. Trybek et al. also established a statistical connection between the existence of leukoplakia and smokeless tobacco consumption. Increase in the nonCandida albicans Candida species, as found in our study, was observed by Chakraborty et al.[13], who concluded Candida tropicalis is ubiquitous yeast in Asia, similarly, the study by Sonia Silva et al.[14] concluded that nonCandida albicans Candida species such as Candida glabrata, Candida tropicalis are now commonly found as human pathogens.

The presence of nonCandida albicans Candida species can be explained on the basis of new medical cures, the upsurge in invasive medical measures, the use of wide-ranging spectrum antibiotics, and poor oral hygiene.[15] However, there are significant variations in Candida species studies. Depending on the geographical region and patient group, some are more predominant in nonCandida albicans Candida species while others are more predominant in Candida albicans in certain countries.

Conclusion

The present study indicated Candida prevalence was observed significantly in the study group in comparison with healthy controls indicating increase in Candida in smokeless tobacco users. The prevalence of nonCandida albicans Candida among leukoplakia and tobacco pouch keratosis group supporting that there is pervasiveness of significant nonCandida albicans Candida in relation to salivary flow rate and buffer capacity. Therefore, the choice of antifungal treatment immediately must deal not only with resistance but similarly also with the transformation in predominance from Candida albicans to nonCandida albicans Candida species. Although the numbers of sample in the study were considerably low to conclude such result, numbers of studies should be performed on larger scale in a particular area.

Financial support and sponsorship

Nil.

Conflicts of interest

There is no conflicts of interest.