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Oral Microflora among Oral Cancer Patients Undergoing Radiotherapy in Regional Cancer Center, Indira Gandhi Medical College, Shimla

CC BY-NC-ND 4.0 ? Indian J Med Paediatr Oncol 2019; 40(S 01): S61-S64

DOI: DOI: 10.4103/ijmpo.ijmpo_247_17

Author : Fotedar, Vikas, Ganju, Suniti; Fotedar, Shailee,Thakur, Purnima, Sharma, Aman, Bhardwaj, Vinay

Abstract

Objective:?The objective of this study was to identify the microflora, especially Gram-positive, Gram-negative, and?Candida?species, in patients with oral squamous cell carcinoma during various stages from diagnosis through radiotherapy.?Materials and Methods:?A total of 17 cases with histological diagnosis of squamous cell carcinoma of the oral cavity were enrolled in the study. For each patient, the sample was collected thrice, i.e., at the time of diagnosis (Sample 1), 14th?15th?day (Sample 2), and on the 29th?30th?day of radiotherapy (Sample 3). The swab stick was rolled across the oral mucosa in the cases and was sent immediately to the Department of Microbiology, Indira Gandhi Medical College, Shimla, for processing. The swabs were inoculated on MacConkey agar, blood agar, and Sabouraud dextrose agar. After overnight incubation at 37?C, the organisms were identified by colony characteristics, catalase, coagulase test, Gram staining, and standard biochemical tests.?Results:?Out of 17, there was a loss to follow up in three patients, so after analyzing on 14 patients, we had 12 (85.7%) males and 2 (14.3%) females. The mean age of the population was 47.6% ? 12.2%. We had significantly higher proportion of Gram-positive microorganisms in Sample 1 as compared to Sample 3 and the same proportion of Gram-negative organisms in Sample 1 and Sample 3.?Candida?species was also proportionately higher in Sample 3 as compared to Sample 1.?Conclusion:?There is a shift of oral microflora from Gram-positive to?Candida?species from Sample 1 to Sample 3 and Gram-negative being same in Sample 1 and Sample 3.

Keywords

Candida - Oral cancer - radiotherapy and oral microflora


Publication History

Article published online:
24 May 2021

? 2019. Indian Society of Medical and Paediatric Oncology. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India

Abstract

Objective:?The objective of this study was to identify the microflora, especially Gram-positive, Gram-negative, and?Candida?species, in patients with oral squamous cell carcinoma during various stages from diagnosis through radiotherapy.?Materials and Methods:?A total of 17 cases with histological diagnosis of squamous cell carcinoma of the oral cavity were enrolled in the study. For each patient, the sample was collected thrice, i.e., at the time of diagnosis (Sample 1), 14th?15th?day (Sample 2), and on the 29th?30th?day of radiotherapy (Sample 3). The swab stick was rolled across the oral mucosa in the cases and was sent immediately to the Department of Microbiology, Indira Gandhi Medical College, Shimla, for processing. The swabs were inoculated on MacConkey agar, blood agar, and Sabouraud dextrose agar. After overnight incubation at 37?C, the organisms were identified by colony characteristics, catalase, coagulase test, Gram staining, and standard biochemical tests.?Results:?Out of 17, there was a loss to follow up in three patients, so after analyzing on 14 patients, we had 12 (85.7%) males and 2 (14.3%) females. The mean age of the population was 47.6% ? 12.2%. We had significantly higher proportion of Gram-positive microorganisms in Sample 1 as compared to Sample 3 and the same proportion of Gram-negative organisms in Sample 1 and Sample 3.?Candida?species was also proportionately higher in Sample 3 as compared to Sample 1.?Conclusion:?There is a shift of oral microflora from Gram-positive to?Candida?species from Sample 1 to Sample 3 and Gram-negative being same in Sample 1 and Sample 3.


Keywords

Candida - Oral cancer - radiotherapy and oral microflora

Introduction

Oral and oropharyngeal carcinomas are the sixth most common cancers in the world.[1] In India, oral cancer is one of the most common cancers and constitutes a major public health problem. The incidence rates of cancers of the oral cavity in both males and females in all urban cancer registries of India are among the highest in the world.[2] Oral cancers have a significant impact on the patient?s quality of life because of the functional loss that results with the treatment modalities even with the highest care rendered nowadays.

Oral cancers have a multifaceted etiology. Although smoking, tobacco chewing, and alcohol consumption are widely associated with oral cancers,[1] these traditional risk factors alone fail to explain the changing epidemiological pattern of increasing incidence of oral cancers in many parts of the world.[3]

Most of the oral cancers are very much advanced at presentation and present a formidable challenge with regard to management to the oral surgeon. With the continuing advances in the techniques and modalities of radiation therapy, a large percentage of head-and-neck tumors are treated by radiation alone or by a combination of radiation and surgery. As it is less disfiguring and disabling, radiation therapy is usually the more desirable modality of the treatment for head-and-neck cancer, provided of course that there is a choice of therapy.

Mucositis and salivary gland hypofunction continue to be inevitable outcomes of radiotherapy.[4] It is thought that the incidence and severity of radiotherapy-associated mucositis is caused in part by changes in the oral bacterial microflora. Oral microorganisms, especially Gram-negative, are believed to be involved in the ulceration phase, where they probably intensify the inflammatory process and aggravate or promote the formation of ulcers.[5] Further, ulceration acts as a portal of entry for microorganisms into the bloodstream leading to local and systemic infections.[6] [7]

Several studies have shown abnormal flora in head-and-neck radiotherapy patients before and after treatment.[8] [9] With this background, the present study was conducted to identify the microflora, especially Gram-positive, Gram-negative, and?Candida?species, in patients with oral squamous cell carcinoma during various stages from diagnosis through radiotherapy.


Materials and Methods

The present study was conducted in the Department of Radiation Oncology, Indira Gandhi Medical College, Shimla. The permission to conduct the study was taken from the head of the institute. After obtaining the informed consent from the patients, a total of 17 cases with proven histological diagnosis of squamous cell carcinoma of the oral cavity were enrolled in the study. For each patient, the sample was collected thrice, i.e., at the time of diagnosis (Sample 1), 14th?15th?day (Sample 2), and on the 29th?30th?day of radiotherapy (Sample 3).

The swab stick was rolled across the oral mucosa in the cases and was sent immediately to the Department of Microbiology, Indira Gandhi Medical College, Shimla, for processing. The swabs were inoculated on MacConkey agar, blood agar, and Sabouraud dextrose agar. After overnight incubation at 37?C, the organisms were identified by colony characteristics, catalase, coagulase test, Gram staining, and standard biochemical tests.

Inclusion criteria

Patients diagnosed with histologically proven oral squamous cell carcinoma scheduled for radiotherapy were included in the study.


Exclusion criteria

Patients on antibioticsPatients on corticosteroidPatients with any systemic disease that will bring about a change in the oral microflora were excluded from the study.


Statistical analysis

The data were analyzed by SPSS version 16 (SPSS Inc., Chicago, IL). The test used was Mc Nemar test.?P?value ?.05 was considered statistically significant


Results

The study sample consisted of 17 participants. Out of 17, three were lost to follow up either in Sample 2 or Sample 3 and were thus excluded from the study. Hence, the analysis was done for 14 participants. Out of 14 participants, there were 2 females (14.3%) and 12 males (85.7%). The mean age of the population was 47.6% ? 12.2% with a range of 29?65 years. We had a maximum number of participants in the age group of 56?65 years (35.7%). The most common site of squamous cell carcinoma was buccal mucosa in 8 (57.2%), followed by tongue in 4 (28.6%) and alveolus and lower lip in 1 (7.1%) each [Table 1] and [2].


Table 1

Age- and gender-wise distribution

Age (years)

Number of participants (%)

Gender

n (%)

26-35

3 (21.4)

Male

12 (85.7)

36-45

3 (21.4)

Female

2 (14.3)

46-55

3 (21.4)

56-65

5 (35.8)
Table 2

Distribution of participants according to site involved

Site involved

n (%)

Buccal mucosa

8 (57.2)

Tongue

4 (28.6)

Alveolus

1 (7.1)

Lower lip

1 (7.1)
Table 3

Microorganisms present at various levels of radiotherapy

On comparing the Sample 1 and Sample 2, both had 21.4% of participants with Gram-positive microorganisms. Gram-negative organisms were found in 42.9% in Sample 1 and 35.7% in Sample 2 and?Candida?was found in 21.4% in Sample 1 and 14.2% in Sample 2, but the difference was not statistically significant [Table 4].

Organism

Sample 1 (%)

Sample 2 (%)

Sample 3 (%)

Alpha hemolytic streptococci

1 (7.1)

1 (7.1)

1 (7.1)

Acinetobacter

1 (7.1)

1 (7.1)

0 (0)

C species

2 (14.2)

2 (14.3)

4 (28.5)

Commensals

3 (21.4)

6 (42.9)

4 (28.6)

Cons

1 (7.1)

1 (7.1)

0 (0)

Escherichia coli

4 (28.5)

3 (21.4)

6 (42.8)

Pseudomonas

1 (7.1)

0 (0)

0 (0)

Staphylococcus aureus

1 (7.1)

0 (0)

0 (0)
Table 4

Comparison of microorganisms in various groups

On comparing Sample 1 and Sample 3, Gram-positive organism was found in 21% in Sample 1 and 7.1% in Sample 3, and the difference was statistically significant (P?= 0.047). Gram-negative organisms were found in equal proportions in both Sample 1 and Sample 3.?Candida?species was found in the higher percentage of participants in Sample 3 (35.7%) as compared to Sample 1 (21.4%), but the difference could not reach statistical significance [Table 4].

Discussion

The present study tried to identify the change in oral microflora in patients with oral carcinomas at the time of diagnosis, 15 days after radiotherapy, and 30 days after radiotherapy. The results of the study show that at the time of diagnosis and 15 days after radiotherapy, Gram-positive microflora was found in 21.4% of participants which reduced to 7.1?ter 30 days of radiotherapy. Furthermore, the?Candida?species increased from Sample 1 to Sample 3 though not significantly, so we can say that there was a shift in oral microflora from Gram-positive organisms to?Candida?species. These findings are in accordance with Almst?hl?et al.[7] and Samaranayake?et al.[8] Several factors may contribute to the oral colonization by these microbial species, such as radiation-induced hyposalivation, smoking, alcohol abuse, and poor oral and corporal hygiene standards, which are commonly observed in most patients with head-and-neck cancer.[9] [10] In our study also, 28 (93.3%) participants had a positive history of tobacco use (60% in the form of smoking and 33% in the form of chewing) and 70% had a positive history of alcohol use.

Oral candidal carriage was reported by various authors in healthy as well as in individuals undergoing radiotherapy, the findings of which were consistent with the present study. The prevalence of yeast in the oropharynx of healthy volunteers was reported to be 35% by Cohen?et al.[11] and 38% by Ramirez-Amador?et al. In the present study,?Candida?species was found in 21.4% in Sample 1 and 35.7% in Sample 3 which is lower than the findings of Ramirez-Amador?et al.,[12] who reported that?Candida?prevalence in patients increased from 43% in the first sample to 62% during radiotherapy and finally to 75% at the end of radiotherapy. The positivity could be due to the fact that the patients are often unable to maintain adequate nutritional status and oral hygiene during radiotherapy, in spite of receiving instructions and care.

Gaetti-Jardim?et al.[13] reported a higher proportion of Gram-negative organisms in Sample 3 as compared to Sample 1 which is in contrast to our study where we had the same proportion in Sample 1 and Sample 3, and in Sample 2, there was a slight reduction in Gram-negative microorganisms. Gram-negative bacteria such as?Enterobacter?and?Pseudomonas?are believed to be involved in the ulceration phase, where they probably intensify the inflammatory process and aggravate or promote the formation of ulcers by the release of proteases and endotoxins [11] which interact with host cells to induce the production and secretion of proinflammatory cytokines [14] [15] resulting in mucositis. These enteric microorganisms frequently act as reservoirs for antimicrobial resistance genes, which spread to other microbial populations.[16] Further, ulceration acts as a portal of entry for microorganisms into the bloodstream leading to local and systemic infections.[17]

The limitation of the present study is the small sample size of the study. Hence, more participants are required to increase the reliability of the results, and more detailed studies are necessary to understand the relationship between radiotherapy and alterations in the nature and magnitude of the oral microflora. Better characterization of changes in oral microflora would be obtained using molecular biological techniques. This would help our understanding of the potential role of oral microflora in the development and exacerbation of oral mucositis. Data from such work could be directed toward developing and testing selective antimicrobial therapies for the prevention and management of mucositis during cancer chemotherapy.

Conclusion

In this small-scaled patient study, we tried to investigate microflora at the time of diagnosis, in between radiotherapy, and at the end of radiotherapy. It was seen that there was a significant reduction in Gram-positive microorganisms at the end of radiotherapy as compared to the start of radiotherapy. There was no change in Gram-negative microorganisms at the beginning of radiotherapy and at the end of radiotherapy. Furthermore, there was an increase in proportion of?Candida?species though not statistically significant from the beginning of radiotherapy to the end of radiotherapy.

Conflict of Interest

There are no conflicts of interest.

  • References

Microorganism

Sample 1 (%)

Sample 2 (%)

P value

Sample 1 (%)

Sample 3 (%)

P value

*Statistically significant

Gram-positive

3 (21.4)

3 (21.4)

1.000

3 (21.4)

1 (7.1)

0.047*

Gram-negative

6 (42.9)

5 (35.7)

1.000

6 (42.9)

6 (42.9)

1.000

Candida albicans

3 (21.4)

2 (14.2)

1.000

3 (21.4)

5 (35.7)

0.275
  1. Warnakulasuriya S.?Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009; 45: 309-16
  2. Nair U, Bartsch H, Nair J.?Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: A review of agents and causative mechanisms. Mutagenesis 2004; 19: 251-62
  3. Hooper SJ, Wilson MJ, Crean SJ.?Exploring the link between microorganisms and oral cancer: A systematic review of the literature. Head Neck 2009; 31: 1228-39
  4. Logan RM.?Advances in understanding of toxicities of treatment for head and neck cancer. Oral Oncol 2009; 45: 844-8
  5. Vissink A, Jansma J, Spijkervet FK, Burlage FR, Coppes RP.?Oral sequelae of head and neck radiotherapy. Crit Rev Oral Biol Med 2003; 14: 199-212
  6. Al-Nawas B, Gr?tz KA.?Prospective study of the long term change of the oral flora after radiation therapy. Support Care Cancer 2006; 14: 291-6
  7. Almst?hl A, Wikstr?m M, Fagerberg-Mohlin B.?Microflora in oral ecosystems in subjects with radiation-induced hyposalivation. Oral Dis 2008; 14: 541-9
  8. Samaranayake LP, Robertson AG, MacFarlane TW, Hunter IP, MacFarlane G, Soutar DS. et al.?The effect of chlorhexidine and benzydamine mouthwashes on mucositis induced by therapeutic irradiation. Clin Radiol 1988; 39: 291-4
  9. Epstein JB, Freilich MM, Le ND.?Risk factors for oropharyngeal candidiasis in patients who receive radiation therapy for malignant conditions of the head and neck. Oral Surg Oral Med Oral Pathol 1993; 76: 169-74
  10. Hasturk H, Nunn M, Warbington M, Van Dyke TE.?Efficacy of a fluoridated hydrogen peroxide-based mouthrinse for the treatment of gingivitis: A randomized clinical trial. J Periodontol 2004; 75: 57-65
  11. Cohen R, Roth FJ, Delgado E, Ahearn DG, Kalser MH.?Fungal flora of the normal human small and large intestine. N Engl J Med 1969; 280: 638-41
  12. Ramirez-Amador V, Silverman Jr. S, Mayer P, Tyler M, Quivey J.?Candidal colonization and oral candidiasis in patients undergoing oral and pharyngeal radiation therapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997; 84: 149-53
  13. Gaetti-Jardim EJ, Ciesielski FI, de Sousa FR, Nwaokorie F, Schweitzer CM, Avila-Campos MJ. et al.?Occurrence of yeasts, pseudomonads and enteric bacteria in the oral cavity of patients undergoing head and neck radiotherapy. Braz J Microbiol 2011; 42: 1047-55
  14. Laheij AM, de Soet JJ, von dem Borne PA, Kuijper EJ, Kraneveld EA, van Loveren C. et al.?Oral bacteria and yeasts in relationship to oral ulcerations in hematopoietic stem cell transplant recipients. Support Care Cancer 2012; 20: 3231-40
  15. K?stler WJ, Hejna M, Wenzel C, Zielinski CC.?Oral mucositis complicating chemotherapy and/or radiotherapy: Options for prevention and treatment. CA Cancer J Clin 2001; 51: 290-315
  16. Nape?as JJ, Brennan MT, Bahrani-Mougeot FK, Fox PC, Lockhart PB.?Relationship between mucositis and changes in oral microflora during cancer chemotherapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 103: 48-59
  17. Gon?alves MO, Coutinho-Filho WP, Pimenta FP, Pereira GA, Pereira JA, Mattos-Guaraldi AL. et al.?Periodontal disease as reservoir for multi-resistant and hydrolytic enterobacterial species. Lett Appl Microbiol 2007; 44: 488-94

Address for correspondence

Dr. Vikas Fotedar
Department of Radiation Oncology, Regional Cancer Center, Indira Gandhi Medical College
Shimla, Himachal Pradesh
India???

Publication History

Article published online:
24 May 2021

? 2019. Indian Society of Medical and Paediatric Oncology. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India


  • References

  1. Warnakulasuriya S.?Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009; 45: 309-16
  2. Nair U, Bartsch H, Nair J.?Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: A review of agents and causative mechanisms. Mutagenesis 2004; 19: 251-62
  3. Hooper SJ, Wilson MJ, Crean SJ.?Exploring the link between microorganisms and oral cancer: A systematic review of the literature. Head Neck 2009; 31: 1228-39
  4. Logan RM.?Advances in understanding of toxicities of treatment for head and neck cancer. Oral Oncol 2009; 45: 844-8
  5. Vissink A, Jansma J, Spijkervet FK, Burlage FR, Coppes RP.?Oral sequelae of head and neck radiotherapy. Crit Rev Oral Biol Med 2003; 14: 199-212
  6. Al-Nawas B, Gr?tz KA.?Prospective study of the long term change of the oral flora after radiation therapy. Support Care Cancer 2006; 14: 291-6
  7. Almst?hl A, Wikstr?m M, Fagerberg-Mohlin B.?Microflora in oral ecosystems in subjects with radiation-induced hyposalivation. Oral Dis 2008; 14: 541-9
  8. Samaranayake LP, Robertson AG, MacFarlane TW, Hunter IP, MacFarlane G, Soutar DS. et al.?The effect of chlorhexidine and benzydamine mouthwashes on mucositis induced by therapeutic irradiation. Clin Radiol 1988; 39: 291-4
  9. Epstein JB, Freilich MM, Le ND.?Risk factors for oropharyngeal candidiasis in patients who receive radiation therapy for malignant conditions of the head and neck. Oral Surg Oral Med Oral Pathol 1993; 76: 169-74
  10. Hasturk H, Nunn M, Warbington M, Van Dyke TE.?Efficacy of a fluoridated hydrogen peroxide-based mouthrinse for the treatment of gingivitis: A randomized clinical trial. J Periodontol 2004; 75: 57-65
  11. Cohen R, Roth FJ, Delgado E, Ahearn DG, Kalser MH.?Fungal flora of the normal human small and large intestine. N Engl J Med 1969; 280: 638-41
  12. Ramirez-Amador V, Silverman Jr. S, Mayer P, Tyler M, Quivey J.?Candidal colonization and oral candidiasis in patients undergoing oral and pharyngeal radiation therapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997; 84: 149-53
  13. Gaetti-Jardim EJ, Ciesielski FI, de Sousa FR, Nwaokorie F, Schweitzer CM, Avila-Campos MJ. et al.?Occurrence of yeasts, pseudomonads and enteric bacteria in the oral cavity of patients undergoing head and neck radiotherapy. Braz J Microbiol 2011; 42: 1047-55
  14. Laheij AM, de Soet JJ, von dem Borne PA, Kuijper EJ, Kraneveld EA, van Loveren C. et al.?Oral bacteria and yeasts in relationship to oral ulcerations in hematopoietic stem cell transplant recipients. Support Care Cancer 2012; 20: 3231-40
  15. K?stler WJ, Hejna M, Wenzel C, Zielinski CC.?Oral mucositis complicating chemotherapy and/or radiotherapy: Options for prevention and treatment. CA Cancer J Clin 2001; 51: 290-315
  16. Nape?as JJ, Brennan MT, Bahrani-Mougeot FK, Fox PC, Lockhart PB.?Relationship between mucositis and changes in oral microflora during cancer chemotherapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 103: 48-59
  17. Gon?alves MO, Coutinho-Filho WP, Pimenta FP, Pereira GA, Pereira JA, Mattos-Guaraldi AL. et al.?Periodontal disease as reservoir for multi-resistant and hydrolytic enterobacterial species. Lett Appl Microbiol 2007; 44: 488-94

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