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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 12  |  Issue : 5  |  Page : 109-113  

Evaluation of alteration in oral microbial flora pre- and postradiation therapy in patients with head and neck cancer


1 Department of Oral Pathology, Chettinad Dental College and Research Institute, Chennai, Tamil Nadu, India
2 Department of Public Health Dentistry, SRM Dental College, Chennai, Tamil Nadu, India
3 Department of Oral Medicine and Radiology, SRM Dental College, Chennai, Tamil Nadu, India
4 Department of Prosthodontics, Meenakshi Ammal Dental College and Hospital, Chennai, Tamil Nadu, India
5 Department of Oral Medicine and Radiology, Chettinad Dental College and Research Institute, Chennai, Tamil Nadu, India

Date of Submission25-Jan-2020
Date of Acceptance02-Apr-2020
Date of Web Publication28-Aug-2020

Correspondence Address:
Ayanambakkam Hemasundar Harini Priya
Department of Oral Pathology, Chettinad Dental College and Research Institute, Rajiv Gandhi Salai, Kanchipuram District., Kelambakkam, Chennai 603103, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.JPBS_38_20

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   Abstract 

Introduction: For decades now, head and neck cancer (HNC) remains to be one of the deadliest; per se the treatment for the same has been evolving for the past years. Radiation therapy (RT) has been one among the various treatment modalities adopted to treat HNC. The fact that RT can affect the quality of life in these patients cannot be denied. In patients with HNC, these can bring about a wide array of lesions in oral cavity and its associated structures as these areas are invariably affected due to exposure to radiation as such. These include alteration in salivary parameters, changes in microbial flora, and occurrence of radiation caries. Aim: In our study we aimed at assessing the alteration in oral microbial flora inclusive of Streptococcus mutans and Lactobacillus colony count before and after RT. Materials and Methods: Saliva and plaque samples were collected from patients with HNC, who were prescribed to undergo intensity-modulated radiation therapy (IMRT) amounting to a mean radiation dosage of 60 Gy units. The aforementioned samples were collected at three different intervals, before, immediately after, and 6 months after RT. The samples were cultured in selective media in three different dilutions (1:10, 1:100, and 1:1000). Following with colony counting is carried out using a standardized colony counter. Conclusion: The results showed a significant alteration in microbial flora pre- and post-RT. Individual parameters do not show a single stand in causing these changes. Moreover, these changes seem to have earned a multifactorial contribution.

Keywords: Head and neck cancer, Lactobacilli species, radiation therapy, saliva, Streptococcus mutans


How to cite this article:
Priya AH, Rajmohan, Arun Kumar HP, Akash Raj S, Archana S, Venkatanarasu B. Evaluation of alteration in oral microbial flora pre- and postradiation therapy in patients with head and neck cancer. J Pharm Bioall Sci 2020;12, Suppl S1:109-13

How to cite this URL:
Priya AH, Rajmohan, Arun Kumar HP, Akash Raj S, Archana S, Venkatanarasu B. Evaluation of alteration in oral microbial flora pre- and postradiation therapy in patients with head and neck cancer. J Pharm Bioall Sci [serial online] 2020 [cited 2020 Sep 19];12, Suppl S1:109-13. Available from: http://www.jpbsonline.org/text.asp?2020/12/5/109/292865




   Introduction Top


Head and neck cancer (HNC) represents ninth most common malignancy on the planet with high death rate; these sicknesses tend to influence the developing nations in high percentages.[1] These groups of disorders have a tendency to originate overwhelmingly from the epithelium covering the oral mucosal surface, nasal cavity, and pharyngeal mucosa mainly the hypopharynx, larynx, and the upper aero digestive tract.[2],[3]

The event of these neoplastic lesions is attributed dominatingly to the use of tobacco both in smoking and smokeless forms, liquor use, diet, few viral infections inclusive of human papillomavirus, preexisting systemic illness, and genetic causes.[4],[5]

The principle aim of malignancy treatment was to treat the condition as early as possible to avoid recurrence and metastasis of primary lesion.[6] The treatment ranges from surgery, chemotherapy to radiation therapy (RT).[7] RT has evolved to an unimaginable extent; it dates back to 1900s.[2] The principle advantage of RT compared to other treatment approaches is that it can provide long-term control of local or locoregional cancer without removal of large volume of tissue along with preservation of the adjacent normal tissues.[3] Although RT has proved to be significantly associated with improved survival rate of patients with HNCs, the quality of life is still at stake. The long-term effects of RT pertaining to head and neck radiation oncology is inclusive of radiation caries (RC), xerostomia, radiation-induced mucositis, salivary gland dysfunction, craniofacial disturbances, nerve damage, osteoradionecrosis, and trismus.[8] These changes can be due to direct or indirect effect of RT. RC remains as an important long-term effect of RT, which in turn can also be attributed to altered oral microbial flora.


   Materials and Methods Top


The study was approved by the ethical board of SRM University. The samples were recruited from two private hospitals after obtaining due consent from the hospital authorities and the patients. Strict inclusion and exclusion criteria were followed so as to avoid errors from adjuvant factors.

Inclusion criteria

The inclusion criteria of the study included the following:

  • Age group: 18 years of age or older


  • Any gender


  • Affected by HNC excluding lymphoma


  • Four posterior teeth and four anterior teeth should be present


  • No anticipated chemotherapy during the course


  • Patients with HNC undergoing intensity modulated radiation therapy (IMRT) of dosage 60–66 Gy units only for 8 weeks.


  • Exclusion criteria

    The exclusion criteria of the study included the following:

  • Any systemic illness causing xerostomia


  • Patients with removable or fixed appliances


  • Receiving antibiotics during therapy or within 3 months before the study


  • Edentulous patients


  • Sample selection



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    The samples were categorized into three groups

    Group I: Before RT

    Group II: After RT

    Group III: Six months after RT

    Sample collection

    Unstimulated saliva samples were collected under suitable aseptic conditions using routine protocol.

    Unstimulated saliva was collected in sterile cups from patients with HNC.



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    pH of the saliva was estimated using pH meter at the chair side.



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    Saliva was then transferred to a graduated sterile saliva collecting tube using a 5-mL syringe and salivary flow rate was estimated.



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    The saliva sample was then centrifuged.



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    Supernatant was transferred into aliquot’s using micropipette and sent for microbial analysis.

    Microorganisms are cultured using selective media to determine to isolate a selective organism as such and to determine its abundance in the sample being tested. The following two specific medias are used:

  • Mitis salivarius-bacitracin (MSB) agar for Streptococcus mutans.


  • Lactobacillus MRS agar (De Man, Rogosa and Sharpe agar) for Lactobacilli species


  • Duplicate samples were plated as 1 in 10 (1/10), 1 in 100 (1/100), 1 in 1000 (1/1000) dilutions, and spread on the surface of the medium. Surface plating method is usually adopted. The plates were then examined under a Digital Colony Counter. Paired t test was performed. The results were then tabulated.


       Results Top


    Paired t test was performed to compare and estimate the difference between the groups.

    [Table 1][Table 2][Table 3] and [Graph 1] show evidence of statistically significant difference between Groups I and II, Groups I and III, and Groups II and III.
    Table 1: Comparison of Streptococcus mutans (cfu/μL) between Groups I and II

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    Table 2: Comparison of Streptococcus mutans (cfu/μL) between Groups I and III

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    Table 3: Comparison of Streptococcus mutans (cfu/μL) between Groups II and III

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    Graph 1: Streptococcus mutans colony count (in cfu/μL) in Groups I, II, and III

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       Discussion Top


    HNCs are often treated with RT, a precise technique that uses ionizing radiation. RT exerts therapeutic effect by scrupulously impairing the genetic material of susceptible malignant cells, either directly or via the release of free radicals by various mechanisms, thereby resulting in cell death.[9] RC can occur by two mechanisms, pertaining to etiology and pathogenesis: One is because of the direct effect of radiation so imparted on the tooth lying in the field of RT and the other is by indirect mechanism, attributed to the alteration in the salivary parameters (flow rate and pH), profound changes in the oral microbial flora (majorly S. mutans), and lack of maintenance of oral hygiene in radiotherapy patients.[10]

    In this study, we aimed at analyzing the alteration in oral microbial flora pertaining to RC, which includes analysis of S. mutans and Lactobacilli colony count. Thus, our study showed a significant increase in S. mutans colony count before and after RT, before, and 6 months after RT. The elevation in colony count between Groups II and III, although statistically significant, was not as remarkable as former, which was in accordance with that of Brown et al.[11] The results so obtained were log10 count in the range of 2–3 before RT, which escalated to log10 count in the range of 3–4 after RT. On follow-up, the values further rose up to log10 count of more than 5 at the end of 3 months. Al-Nawas et al.,[12] found that the mean colony-forming units (CFUs) were approximately 630 (×1000 cfu/mL) before RT, 920 (×1000 cfu/mL) immediately after RT, and in the range of 1000 (×1000 cfu/mL) 6 months after RT.

    Meng et al.[13] have also showed similar results where the mean value before RT was log CFU 6.41 ± 0.81(607.89 ± 2.24 cfu/μL), immediately after RT was log CFU 6.76 ± 0.62 (862.64 ± 1.86 cfu/μL), and 6 months after RT was log CFU 7.68 ± 0.70 (1096.63 ± 2.18). The minor variation in CFUs among the studies might be attributed to the use of different agar medium for culturing the species.

    Our study results were in discordance with those of the study conducted by Schuurhuis et al.,[14] wherein the study was conducted to compare the oral microbial flora on a long-term basis following RT using oral lavages. The results were in contrast to our study, in which the amount of S. mutans before RT was approximately 60%–70%, which reduced to 55%–60% after RT, and which further declined to 50%–60% at the end of 6 months after RT.

    Similar trend was observed with estimation of Lactobacilli colony count as evident in [Table 4][Table 5][Table 6] and [Graph 2], wherein there was a significant increase in Lactobacillus colony count before and after RT, before, and 6 months after RT. The rise in colony count between Groups II and III, although statistically significant, was not as remarkable as the other two.
    Table 4: Comparison of Lactobacillus species (cfu/μL) between Groups I and II

    Click here to view
    Table 5: Comparison of Lactobacillus species (cfu/μL) between Groups I and III

    Click here to view
    Table 6: Comparison of Lactobacillus species (cfu/μL) between Groups II and III

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    Graph 2: Lactobacilluscolony count (in cfu/μL) in Group I, II, and III

    Click here to view


    The results of our study were in accordance with those of the study conducted by Brown et al.,[11] who found that the mean CFUs before RT were log 10 count in the range 1–2 before the start of RT, which escalated to log 10 count in the range 3–4 immediately after RT, raising to log 10 count in the range 4–5 3 months after RT. In a study conducted by Al-Nawas et al.,[12] the mean CFUs were approximately 575 (×1000 cfu/mL) before RT, approximately 600 (×1000 cfu/mL) immediately after RT, and approximately 920 (×1000 cfu/mL) 6 months after RT. Vuotila et al.[15] also conducted a similar study to analyze the salivary microbial alteration before and after RT. The results of the study showed a significant rise in Lactobacillus levels after RT when compared to the values obtained before RT. The results of the study conducted by Schuurhuis et al.[14] were found to be complex in nature, wherein the study was conducted to compare the oral microbial flora on a long-term basis following RT using oral lavages. The amount of Lactobacillus before RT was approximately 70%–80%, which increased to 80%–90% after RT, which was in accordance with our study, but at the end of 6 months after RT the levels declined 50%–60%, which was in contrast to our results. The increase in Lactobacillus level after RT is mainly due to the effect of radiation, which leads to release of reactive oxygen species, thereby creating an acidic environment, in which the organisms multiply faster.[16] The significant fall in Lactobacillus count, 6 months after RT, might be due to two reasons: one is the samples used, where they have used oral lavages instead of saliva. Second, in this study the patients were subjected to an intensive oral care protocol and their oral hygiene improved substantially during follow-up, whereas in our study such protocol was not adopted.[14]


       Conclusion Top


    HNCs are more often treated by RT. Despite the fact that RT assumes a vital role in decimating tumor cells, its destructive symptoms cannot be ignored. RC is one such reaction, which happens on a long-run post-RT. Despite the fact that different investigations examined the immediate and aberrant impact of RT on tooth and its related structures, none were ended up being fruitful in defining a preventive measure to be embraced pre- and post-RT. In our study, we showed a significant alteration in oral microbial flora pertaining to dental caries, thereby suggesting use of antibacterial mouth rinses pre- and post-completion of RT on a long-term basis.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.



     
       References Top

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    2.
    Bernier J, Hall EJ, Giaccia A Radiation oncology: a century of achievements. Nat Rev Cancer 2004;4:737-47.  Back to cited text no. 2
        
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    Do KH General principles of radiation protection in fields of diagnostic medical exposure. J Kor Med Sci 2016;31:S6-9.  Back to cited text no. 3
        
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    Weichselbaum RR, Liang H, Deng L, Fu YX Radiotherapy and immunotherapy: a beneficial liaison? Nat Rev Clin Oncol 2017;14:365-79.  Back to cited text no. 4
        
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    Seyfried TN, Huysentruyt LC On the origin of cancer metastasis. Crit Rev Oncogen 2013;18:43.  Back to cited text no. 6
        
    7.
    Yadav K, Prakash S Dental caries: a review. Asian J Biomed Pharm Sci 2016;53:1-7.  Back to cited text no. 7
        
    8.
    Tolentino ES, Centurion BS, Ferreira LH, Souza AP, Damante JH, Rubira-Bullen IR Oral adverse effects of head and neck radiotherapy: literature review and suggestion of a clinical oral care guideline for irradiated patients. J Appl Oral Sci 2011;19:448-54.  Back to cited text no. 8
        
    9.
    Beech N, Robinson S, Porceddu S, Batstone M Dental management of patients irradiated for head and neck cancer. Aust Dent J 2014;59:20-8.  Back to cited text no. 9
        
    10.
    Thiel HJ Radiation caries. Radiobiol Radiother 1989;30:193-211.  Back to cited text no. 10
        
    11.
    Brown LR, Dreizen S, Handler S, Johnston DA Effect of radiation-induced xerostomia on human oral microflora. J Dent Res 1975;54:740-50.  Back to cited text no. 11
        
    12.
    Al-Nawas B, Grötz KA Prospective study of the long term change of the oral flora after radiation therapy. Support Care Cancer 2006;14:291-6.  Back to cited text no. 12
        
    13.
    Meng L, Liu J, Peng B, Fan M, Nie M, Chen Z, et al. The persistence of Streptococcus mutans in nasopharyngeal carcinoma patients after radiotherapy. Caries Res 2005;39:484-9.  Back to cited text no. 13
        
    14.
    Schuurhuis JM, Stokman MA, Witjes MJ, Langendijk JA, van Winkelhoff AJ, Vissink A, et al. Head and neck intensity modulated radiation therapy leads to an increase of opportunistic oral pathogens. Oral Oncol 2016;58:32-40.  Back to cited text no. 14
        
    15.
    Vuotila T, Ylikontiola L, Sorsa T, Luoto H, Hanemaaijer R, Salo T, et al. The relationship between MMPs and pH in whole saliva of radiated head and neck cancer patients. J Oral Pathol Med 2002;31:329-38.  Back to cited text no. 15
        
    16.
    Ramya AS, Uppala D, Majumdar S, Surekha Ch, Deepak KG Are salivary amylase and pH: prognostic indicators of cancers? J Oral Biol Craniofac Res 2015;5:81-5.  Back to cited text no. 16
        


        Figures

      [Graph 1], [Graph 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
     
     
        Tables

      [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



     

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