Comparative Study about the Ecacy of Low Level Laser Therapy and Curcumin Antimicrobial Photodynamic Therapy as a Coadjuvant Treatment of Oral Mucositis in Oncologic Patients:Antimicrobial, Analgesic and Degree Alteration Effect.

INTRODUCTION: As conventional treatments currently available for mucositis are not considerably effective,there is a need to implement an adjuvant protocol for the treatment of oral mucositis in patients undergoing radiotherapy and chemotherapy. PURPOSE: To evaluate the effect of Low Level Laser Therapy and Antimicrobial Photodynamic Therapy mediated by curcumin and blue LED as an adjunct treatment of oral mucositis foroncology patients using chemotherapy and or radiotherapy. METHODS: Clinical, randomized study, in a single location, in an oncology service of a general hospital, with a total of 30 patients (over 18 years old) with stable oral mucosa lesions in the process of Chemotherapy and or Radiotherapy. The patients were divided into 3 groups: Control group (treated with nystatin), LLLT group (treated with Low Level Laser Therapy), and the aPDT group (treated with 450 nm blue LED and curcumin photosensitizer). RESULTS: The results showed, by means of intra-group comparisons, that the two experimental treatments promoted yeast reduction of the genus Candida in the last two evaluations (21 days and 30 days), but not in the rst two evaluations (7 days and 14 days). The intra-group comparisons showed that the Control and aPDT group showed a signicant difference in the degree of mucositis over the four evaluations performed, with the results pointing out that the mucositis worsened in the control group from the 14th day, while reduced in the aPDT group from the 21st day of treatment. CONCLUSION: A reduction in the degree of mucositis and pain score was observed in the LLLT and aPDT groups, with the aPDT group standing out when presenting early clinical improvement in relation to the LLLT group and the control group, thus emphasizing its effectiveness within the desired aspects. Regarding the antimicrobial effect, aPDT showed a greater reduction of yeasts of the genus Candida in the tested parameters.


Introduction
Cancer treatment is performed through surgery, radiotherapy (RT), chemotherapy (CT), bone marrow transplantation, or even by combining more than one modality [1]. Oral mucositis is a frequent side effect, resulting from chemotherapy and radiotherapy in the treatment of cancer, associated with pain and characterized by in ammation and ulceration of the oral and gastrointestinal mucosa, making it di cult to swallow solid foods, sometimes liquids, also limiting speech and chewing [2][3][4][5]. This condition exposes the individual to generalized infections by opportunistic microorganisms and still usually triggers malnutrition and dehydration with systemic weakness, culminating in the reduction or interruption of antineoplastic therapy [2][3][4][5]. Other factors such as alcoholism and smoking, associated with chemotherapy and/or radiotherapy, fungal infections and poor oral hygiene can increase or worsen the condition [4,6,7].
There are no universally de ned treatment protocols for oral mucositis. Many procedures have been and are used, such as oral hygiene care, use of oral rinses, analgesics, antibiotics, cryotherapy, local anesthetics, growth factors and cytokines, anti-in ammatory agents, among others [8].
It is observed that Low Level Laser Therapy (LLLT) reduces pain, in ammation and edema, promotes healing of deeper tissues and nerves, and has good results for the treatment of oral mucositis in cancer patients, associated with the photobiomodulation process 9 . Another therapy widely usedis the Antimicrobial Photodynamic Therapy (aPDT) based on the activation of photosensitizing agents with a light at a speci c wavelength 1 . The light (laser or LED) activates a photosensitizer that is applied to the target, thus, its toxic effect is explained in two ways: by the process of redox and by the formation of singlet oxygen [4,5,[9][10][11][12]. Among the different types of photosensitive agents used in aPDT, curcumin and curcuminoids have been gaining space in the research elds. These compounds are derived from turmeric, a natural yellow pigment that consists of a mixture of three curcuminoids: curcumin, demetoxicurmine and bis-demetoxicurcumin. In addition to their use as a photosensitizing agent in aPDT, these compounds have pharmacological properties such as: anti-in ammatory, antioxidant, anticancer, antibacterial and antifungal [13].
Thus, in the present study, we proposed to compare the effectiveness of LLLT, aPDT mediated by curcumin and blue light as an adjunct to the treatment of cancer patients using chemotherapy and or radiotherapy with oral mucositis. In addition, this work also aimed to contribute to the knowledge about the clinical characteristics of patients in the studied sample, to evaluate the effectiveness of aPDT mediated by curcumin and blue light in the elimination of yeast of the genus Candida in saliva samples, and to evaluate the effectiveness ofLLLT in the clinical improvement of ulcers in patients with oral mucositis.

Methods
Ethical aspects and study location

Population
The population chosen for this study was composed of cancer patients,submitted to chemotherapy and/or radiotherapy, who seek the Dentistry service of HGVC. The sample calculation showed the need for a sample ofthirty patients with a 95% con dence interval and a sampling error of 5%.
The following inclusion criteria were used for the research: 1) be 18 years or older; 2) have stable lesions in the process of CT and/or RT; 3) Consent to participate in the research by signing the Informed Consent Form. The exclusion criteria were: 1) Patients who were receiving medication for the treatment and prevention of mucositis; 2) Patients unable to comply with the treatment procedure or the oral hygiene protocol.

Statistical Analysis
For the descriptive analysis of the data, the mean, standard deviation and absolute and relative frequencies of the groups were used. Data normality was tested using the ShapiroWilk test and homoscedasticity using Levene's test. Comparisons of demographic and clinical characteristics between experimental groups were made using Fisher's exact test (for sex) and one-way analysis of variance (oneway ANOVA). The immediate effect of treatments on the yeast response of the genus Candida was assessed using the Wilcoxon and t-Student tests for paired samples (for intra-group comparison) and the Mann-Whitney and t-Student tests for independent samples (for intergroup comparison). To verify the late effect of treatments on the yeast response of the genus Candida, mucositis degree and pain, nonparametric statistic testswere used: Friedman test for intra-group comparisons (with comparisons between pairs being tested by the Wilcoxon test) and Kruskal-Wallis test for intergroup comparisons (with comparisons between pairs being tested by the Mann-Whitney test). The level of signi cance adopted in all analyzes was 5% (α = 0.05). The data were tabulated and analyzed using IBM SPSS Statistics for Windows (IBM SPSS. 21.0, 2012, Armonk, NY: IBM Corp.).

Patient Follow-up Care, Safety, and Outcome Measures
The patients included in the study had the characteristics collected: sex, age, type of cancer, location of initial cancer, type of treatment, number of lesions, location of the mucositis according to the anatomical region of the oral mucosa, and respective degree of mucositis(according to World Health Organization WHO: 1-burning, erythema;2-erythema, ulcer, solid diet; − 3-con uence of ulcers, liquid diet; 4-oral feeding is not possible, liquid diet). The data were recorded on individual records.
Samples of unstimulated saliva were collected before and after the proposed treatments. The collection was performed with sterile universal collectors for 1 minute and then identi ed in coded form. Inside the laminar ow, in an aseptic environment, the saliva samples were processed through the decimal dilution of 10 − 1 , 10 − 2 and 10 − 3 . 100µL aliquots of the initial samples and dilutions obtained were seeded in duplicate in Petri dishes containing Sabouraud dextrose agar culture medium plus 0.1 mg/mL of chloramphenicol for the growth of yeast species of the genus Candida spp. The plates were incubated at 37ºC for 48 hours in an Oxygen Chemical Demand (BOD) oven. When there was no growth during this period, the samples were left for another ve days at room temperature. After growth, the colonies were examined for morphological characteristics (size, shape, surface, halos and presence of pigments), and the number of colonies forming units per milliliter (CFU/mL) was counted.
The evaluation of the analgesic effect was performed through the application of the Visual Analogic Scale (VAS) for pain. This assessment was carried out immediately before the patients started the proposed treatments and was always repeated after the completion of each procedure throughout the study period.
The assessment of clinical improvement of mucositis lesions was performed after each procedure once a week taking into account a graded classi cation proposed by the WHO.
For the aPDT group, the photosensitizing agent was prepared at the time of use with the amount of 7.5 mg of curcumin (Sigma-Aldrich, St. Louis, USA). The curcumin solution (10 ml) that was sprayed inside the oral cavity remained for 10 minutes for impregnation or pre-irradiation time, with the mouth closed to prevent the incidence of ambient light. After the photosensitizing agent was impregnated, the oral cavity

Results
In the rst stage of this study, patients had information collected about their health conditions and sociodemographic variables. The analysis of demographic characteristics did not show any signi cant difference between the groups regarding the distribution between sexes and mean age (Table 1). The immediate effect of LLLT and aPDT on theCandida yeast response over different treatment sessions can be seen in Table 2. It was found through intra-group comparisons that the two treatments reduced Candida yeast in both treatments last evaluations (21 days and 30 days), but not in the rst two (7 days and 14 days). However, intergroup comparisons indicated that the average percentage of yeast reduction in the genus Candida after the application of therapies was signi cantly higher in the aPDT group in all four assessments (7, 14, 21 and 30 days). The late effect of treatments on the yeast response of the genus Candida, degree of mucositis and pain are shown in Table 2. It was observed through intra-group comparisons that none of the groups showed a statistical difference in response in the yeast of the genus Candida between the four evaluations performed. However, intergroup comparisons indicated a signi cant difference in the third (21 days) and fourth (30 days) evaluations, with the results pointing out that, compared to the control group, the LLLT group had a lower number of colony-forming units in the last evaluation (30 days) and the aPDT group had the lowest number in the last two evaluations (21 and 30 days). There were no statistical differences between the LLLT and aPDT groups in the bacterial response. The intra-group comparisons showed that the Control and aPDT groups showed a signi cant difference in the degree of mucositis over the four evaluations performed, with the results pointing out that mucositis worsened in the control group from the 14th day, while it decreased in the aPDT group from the 21st day of treatment. Intergroup comparisons indicated statistical difference in the third (21 days) and fourth (30 days) evaluations, with the results showing that, compared to the control group, the LLLT group had a lower degree of mucositis in the last evaluation (30 days) and the aPDT group in the last two evaluations (21 and 30 days). There were no statistical differences between the LLLT and aPDT groups in the degree of mucositis ( Table 3). The results of the intra-group comparisons indicated that all three groups showed a statistical difference in the pain score over the four evaluations performed, with the results demonstrating that in the LLLT group there was pain reduction after 21 days of treatment, while in the aPDT group this effect was observed earlier (14 days); in the control group there was a reduction in pain in the third assessment (21 days), followed by an increase in the last assessment (30 days).
Intergroup comparisons showed a greater analgesic effect of treatments with LLLT and aPDT at the end of the study (30 days), compared to the control group (Table 3).

Discussion
Cancer is a major cause of morbidity and mortality worldwide, with incidence rates that vary widely depending on geographic location, age, sex and race. Low-and middle-income countries have a proportionally higher burden of cancer than high-income countries, but their health systems are not prepared to deal with this problem [14].
For Brazil, it is estimated that 11.200 new cases of oral cavity cancer in men and 3.500 in women are estimated for each year of the 2018-2019 biennium. These numbers correspond to an estimated risk of 10.86 new cases per 100.000 men, ranking fth; and 3.28 for every 100.000 women, being the 12th most common among all cancers 14 . Of these patients, it is common to develop oral side effects as a result of the treatment to which they are submitted, since changes in the oral microbiota, changes in physiological functions and/or important structural changes are inherent to the treatment and end up causing side effects such as: xerostomia, osteoradionecrosis, candidiasis, mucositis, among other several found in the literature [15][16][17].
The most important side effects of antineoplastic therapy in the past are vomiting and decreased immunity due to myelosuppression, however, due to the use of antibiotics and the use of hematopoietic agents, vomiting and immune compromise have been greatly reduced [15][16][17]. However, mucositis has recently emerged as one of the most serious side effects in cancer treatment [4,5]. When oral mucositis develops during cancer treatment, it can lead to dysphagia, pain, changes in taste, vomiting, nausea, decreased food intake, fatigue and weight loss, in addition, discontinuation of therapy due to mucositis can lead to an extension of the period of the same, which can affect the result obtained or in the deterioration of the quality of life [4,5]. The purpose of rehabilitation is to restore the patient to normal life, promoting the recovery of physical, emotional, mental and social function [18].
When analyzing the results achieved, it is observed that the two treatments reduced Candida yeast in the last two evaluations (21 days and 30 days), but not in the rst two (7 days and 14 days). However, intergroup comparisons indicated that the average percentage of yeast reduction of the genus Candida after the application of therapies was signi cantly higher in the aPDT group in all four assessments (7,

14, 21 and 30 days).
Regarding the antimicrobial e cacy of aPDT, many studies have shown promising results, often reaching inactivation rates of more than 5 log10 CFU, which is understood as a disinfectant effect according to infection control guidelines. Data suggest that photodynamic therapy is potentially effective against bacterial, viral, fungal and protozoal infections [19][20][21][22]. The in vitro mechanism of action described in some studies shows that this therapy induces the generation of reactive oxygen (ROS) and nitrogen species, which effectively damage a variety of fungal cell structures and induce cell death [23][24][25].
The intra-group comparisons demonstrated that the control and aPDT groups showed a signi cant difference in the degree of mucositis over the four evaluations carried out, with the results pointing out that the mucositis worsened in the control group from the 14th day, while reduced in the aPDT group from the 21th day of treatment. Intergroup comparisons indicated statistical difference in the third (21 days) and fourth (30 days) evaluations, with the results showing that, compared to the control group, the LLLT group had a lower degree of mucositis in the last evaluation (30 days) and the aPDT group in the last two evaluations (21 and 30 days). No statistical differences were observed between the LLLT and aPDT groups in the degree of mucositis, demonstrating that both therapies are effective in the treatment of mucositis.
A Systematic Review and Meta-Analysis conducted in 2014 demonstrated that prophylactic LLLT reduces the overall risk of severe mucositis and other measures of mucositis severity, including the duration of severe mucositis in cancer patients and those undergoing aPDT [13]. Numerous studies carried out by scientists have shown that LLLT normalizes microcirculation, activates endothelocytes, stimulating their functional activity by dilating and opening reserve capillaries, providing oxygen access to epithelial cells and promoting activation of cell metabolism [26]. Different studies have described a decrease in the incidence and severity of mucositis, apparently due to accelerated regeneration and affected tissue healing, thereby reducing in ammation and pain [9]. The application of LLLT accelerates oral reepithelization, favorably in uencing the results of oral mucositis in patients undergoing hematopoietic stem cell transplantation and myeloablative chemotherapy [7].
The results of the intra-group comparisons indicated that all three groups showed a statistical difference in the pain score over the four evaluations performed, with the results demonstrating that in the LLLT group there was pain reduction after 21 days of treatment, while in the aPDT group this effect was observed earlier (14 days); in the control group there was a reduction in pain in the third assessment (21 days), followed by an increase in the last assessment (30 days). Intergroup comparisons showed a greater analgesic effect of treatments with LLLT and aPDT at the end of the study (30 days), compared to the control group. The results presented here clearly demonstrate the effectiveness of these treatment modalities, proving to be a valid option for improving patients' quality of life.
Pain associated with mucositis often impairs functional status, this painful side effect reduces quality of life and often requires narcotic analgesia, enteral or parenteral nutrition at additional costs [27].
It is noteworthy that, to date, a large number of reliable studies, which prove the effectiveness of LLLT in the treatment of mucositis after chemotherapy, have been published, and they report a reduction in the likelihood of complications, in the severity of the disease and in the pain level. The preventive use of LLLT does not allow oral mucositis to develop, signi cantly reducing the cost of maintaining patients in the hospital and the total cost of treatment, the high e ciency of LLLT being explained by numerous mechanisms of laser biomodulation [26].

Conclusion
aPDT mediated by curcumin and LLLT are a non-invasive modality for the prevention and management of oral mucositis with some advantages such as antimicrobial effect, analgesic effect and reduction of in ammation.
A reduction in the degree of mucositis and pain score was observed in the LLLT and aPDT groups, with the aPDT group standing out when presenting early clinical improvement in relation to the LLLT group and the control group, thus emphasizing its effectiveness within the desired aspects. However, in aPDT mediated by curcumin when compared with LLLT, a greater reduction of Candida yeasts was noticed. In addition, the average uency of 20.1 J/cm 2 irradiated by LED associated with curcumin was effective in lethal sensitization of yeasts of the genus Candida.