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• Use of photodynamic therapy is suggested for decontamination of dental implants after implantation. • Photodynamic therapy can reduce or eliminate the presence of biofilms. • The report summarizes in vitro studies comparing photodynamic therapy with use of chlorhexidine. Photodynamic therapy may be effective with fewer adverse effects. In vivo studies will be needed to establish safety and efficacy. The aim of this study was to investigate the effects of antimicrobial photodynamic therapy (PDT) using 635 nm diode laser irradiation with an energy density of 6 to 30 J/cm2 and toluidine blue O (TBO) as a photosensitizer on the viability of Aggregatibacter actinomycetemcomitans attached to the surface of titanium implants. Titanium implants contaminated with A. actinomycetemcomitans were treated with TBO alone or in combination with different exposure parameters (light doses of 6 - 30 J/cm2 at 635 nm) and 0.2 % chlorhexidine (CHX). After treatment, colony forming units (CFUs)/ml were determined to assess PDT efficacy. The structure of the biofilm of A. actinomycetemcomitans was analyzed by field emission scanning electron microscopy (FESEM). Under optimal conditions, the colony count was reduced by ∼90 %. Treatment with CHX was somewhat more effective (colony formation was reduced by ∼95 %), but this agent has adverse effects that can be avoided with PDT. This study confirms the efficacy of PDT against A. actinomycetemcomitans depending on the light dose. Treatment with TBO + 635 nm diode laser has an effect that may be equivalent to that of CHX, but perhaps with fewer adverse effects. [ABSTRACT FROM AUTHOR]

• ALA-PDT is a minimally invasive and effective treatment for actinic cheilitis. • Autofluorescence imaging and toluidine blue are alternative noninvasive modalities to visualize lesions and decide the treatment area. • Aneuploidy with DNA image cytometry has monitoring and predictive values in follow-up of actinic cheilitis. • Non-invasive auxiliary tests assisted ALA-PDT is attractive and well-tolerated and may have synergistic effects against actinic cheilitis. Actinic cheilitis (AC) is recognized as the most common precursor lesion of squamous cell carcinoma (SCC) of the lip, with a higher risk of invasiveness and metastasis. Early accurate diagnosis and appropriate therapy are essential to prevent carcinogenesis and progression of AC. Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT), a non-surgical and minimally invasive modality, has been proposed as an effective treatment for oral potentially malignant diseases (OPMDs) and oral cancers. Herein, we report a 64-year-old female patient with AC on the lower lip who received 3 sessions of ALA-PDT with an interval of 1 week. Multiple noninvasive auxiliary tests including autofluorescence imaging, toluidine blue staining, and aneuploidy with DNA image cytometry (DNA-ICM) using brushing from screening through diagnosis, treatment, and follow-up. The patient successfully showed a complete response with no adverse effects and no evidence of recurrence at the 20-month follow-up. Noninvasive auxiliary tests assisted PDT is attractive and well-tolerated and may have synergistic effects against AC. [ABSTRACT FROM AUTHOR]

• Photodynamic therapy is believed to be a promising alternative treatment for Candida infections. • The study evaluated the efficacy of toluidine blue aPDT in the elimination of selected Candida species. • The study confirmed the efficacy of aPDT against C.albicans, C. glabrata and C. krusei. • The outcome of toluidine blue a PDT mostly dependent on the laser parameters and the type of fungus. Photodynamic therapy is believed to be a promising treatment for Candida infections. This study evaluated the efficacy of antimicrobial photodynamic therapy (aPDT) using the 635 nm diode laser light and toluidine blue (TB) in the elimination of selected Candida species cultured on acrylic surface. 108 acrylic plates (Methyl Methacrylate Polymer, routinely used for the production of prosthetic dentures) were placed in three sterile Petri dishes and poured with prepared suspensions of Candida strains: C. albicans , C. glabrata , and C. krusei. After all procedures of fungi incubation, fungal biofilm was visible on the plates' surfaces. The acrylic plates were divided into nine study groups (B) and nine control groups (K) for further experiments. In the study groups, the acrylic plates with fungal biofilm were immersed in TB and afterwards laser irradiation was applicated with different exposure parameters (groups: B1 – 400 mW, 24 J/cm2, 30 s; B2 – 300 mW, 18 J/cm2, 30 s; B3 – 200 mW, 12 J/cm2, 30 s) separately for each Candida species. The control groups contained following parameters: no exposure to laser light or TB, treatment only with TB without laser irradiation, or only laser irradiation without previous immersion in TB. Calculations of colony forming units (CFUs) were conducted by using aCOlyte (Synbiosis). Differences in CFUs were analyzed by the Wilcoxon test. In all study groups, the reduction in CFUs was statistically significant. The differences in CFUs before and after intervention were insignificant. The K3 C.a. control group showed a statistical reduction of Candida albicans after laser irradiation. Our study confirmed the efficacy of aPDT against C. albicans, C. glabrata and C. krusei being dependent on the laser parameters and the type of fungus. The advantage of this study is the validation of aPDT effectiveness in in vitro studies to transpose this data into future clinical trials using photodynamic therapy in the treatment of oral candidiasis. [ABSTRACT FROM AUTHOR]

• The prevalence of peri-implant diseases is increasing. • Antimicrobial photodynamic therapy(aPDT) suggested as an efficient modality for decontamination of implant surface. • aPDT with TBO+635 nm diode laser and ICG+808 nm diode laser decreased the bacterial load on titanium discs. Antimicrobial Photodynamic therapy (aPDT) is a novel modality suggested for treatment of peri-implantitis. This study aimed to assess the effect of aPDT with toluidine blue (TBO) and indocyanine green (ICG) and 635 nm and 808 nm diode laser on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biofilm formed on Laser-Lok titanium discs. Eighty sterile Laser-Lok titanium discs were inoculated with A. actinomycetemcomitans to form biofilm and were randomly divided into 8 groups (n = 10) of control, chlorhexidine (CHX), TBO, ICG, 635 nm diode laser with 220 mW power, 808 nm diode laser with 250 mW power, 100 μg/mL TBO+635 nm diode laser and ICG+808 nm diode laser. Number of colony forming units (CFUs) on the surface of each disc was counted after the intervention. Data were analyzed using the Kruskal-Wallis test. Significant differences were noted in colony count among the eight groups after the intervention (P = 0.001). Pairwise comparisons with adjusted P value test showed that aPDT with TBO+635 nm laser and ICG+808 nm laser caused significant reduction of bacterial biofilm compared to the control group (P = 0.0001). TBO alone caused significant reduction of biofilm compared to the control group (P = 0.004). No other significant differences were noted (P > 0.05). Within the limitations of this study, the results showed that aPDT is a potential modality for decontamination of implant surface and reduction of A. actinomycetemcomitans biofilm in vitro. In this study, aPDT with TBO+635 nm diode laser and ICG+808 nm diode laser decreased the bacterial load on titanium discs. [ABSTRACT FROM AUTHOR]

Highlights • Photodynamic Antimicrobial Chemotherapy (PACT) can decrease growth of yeast. • The transition from mycelium to yeast form can be inhibited by PACT. • PACT can decrease growth of P. brasiliensis in its mycelial form. Abstract Background The thermo-dimorphic fungus Paracoccidioides brasiliensis is the pathogen of Paracoccidioidomycosis, an important public health problem in Latin American with prevalence in Brazil. Photodynamic Antimicrobial Chemotherapy (PACT) is a process that combines a photosensitizer and light, producing reactive oxygen species (ROS) that can promote damages to treated cells. Methods In this work was study the effect of PACT, using Toluidine blue (TBO) on both yeast and mycelial cells of P. brasiliensis. Results It was observed that PACT decreased P. brasiliensis yeast growth, in a dependent manner of both TBO concentrations and fluence. In the presence of TBO 0.005 mg/mL, PACT reduced P. brasiliensis yeast growth in 63, 62 and 86%, using fluences of 20, 30 and 40 J/cm2, respectively. After PACT, ROS production increased 2.80, 4.64 and 7.90 times, in the presence of TBO 0.001, 0.002 and 0.005 mg/mL, respectively. It was observed that after PACT, the cells are predominantly in mycelia form, indicating that mycelial cells irradiated in the presence of TBO, maintained their filamentous form and absence and/or decreased presence of transition structures. Conclusions These results demonstrated the potential of PACT, using TBO to inhibit both yeast and mycelium development of P. brasiliensis. [ABSTRACT FROM AUTHOR]

Antimicrobial Photodynamic Inactivation (a-PDI) is based on the oxidative destruction of biological molecules by reactive oxygen species generated by the photo-excitation of a photosensitive molecule. When a-PDT is performed with the use of mathematical models, the optimal conditions for maximum inactivation are found. Experimental designs allow a multivariate analysis of the experimental parameters. This is usually made using a univariate approach, which demands a large number of experiments, being time and money consuming. This paper presents the use of the response surface methodology for improving the search for the best conditions to reduce E. coli survival levels by a-PDT using methylene blue (MB) and toluidine blue (TB) as photosensitizers and white light. The goal was achieved by analyzing the effects and interactions of the three main parameters involved in the process: incubation time (IT), photosensitizer concentration (C PS ), and light dose (LD). The optimization procedure began with a full 2 3 factorial design, followed by a central composite one, in which the optimal conditions were estimated. For MB, C PS was the most important parameter followed by LD and IT whereas, for TB, the main parameter was LD followed by C PS and IT. Using the estimated optimal conditions for inactivation, MB was able to inactivate 99.999999% CFU mL −1 of E. coli with IT of 28 min, LD of 31 J cm −2 , and C PS of 32 μmol L −1 , while TB required 18 min, 39 J cm −2 , and 37 μmol L −1 . The feasibility of using the response surface methodology with a-PDT was demonstrated, enabling enhanced photoinactivation efficiency and fast results with a minimal number of experiments. [ABSTRACT FROM AUTHOR]

Background Candida albicans is an opportunistic fungus producing both superficial and systemic infections, especially in immunocompromised individuals. It has been demonstrated that C. albicans ability to form biofilms is a crucial process for colonization and virulence. Furthermore, a correlation between the development of drug resistance and biofilm maturation at Candida biofilms has been shown. Photodynamic Antimicrobial Chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a non-toxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using Toluidine Blue O (TBO) on the viability of biofilms produced by C. albicans at different stages of development. Methods In this study, the effects of PACT on both biofilm formation and viability of the biofilm produced by C. albicans were studied. Biofilm formation and viability were determined by a metabolic assay based on the reduction of XTT assay. In addition, the morphology of the biofilm was observed using light microscopy. Results PACT inhibited both biofilm formation and viability of the biofilm produced by C. albicans . Furthermore, PACT was able to decrease the number of both cells and filamentous form present in the biofilm structure. This inhibitory effect was observed in both early and mature biofilms. Conclusions The results obtained in this study demonstrated the potential of PACT (using TBO) as an effective antifungal therapy, including against infections associated with biofilms at different stages of development. [ABSTRACT FROM AUTHOR]

Introduction Many attempts have been made in elimination of bacteria in infected and demineralized dentin to not only provide efficient bactericidal potential, but to have minimal damage for tooth structure. The aim of this study was to assess the temperature change during aPDT with ICG and TBO compared with conventional Diode laser irradiation. Materials and methods 48 premolar teeth which were selected for this study. A class I cavity was drilled in each teeth, with dimensions of 2 mm width, 4 mm length and depth of 2.5–3.5 mm, providing a dentinal wall of approximately 0.5 mm for pulp chamber. Then teeth were randomly allocated in 4 experimental groups (n = 12); Group 1: TBO + LED, Group 2: ICG + Diode Laser, Group 3: Diode laser with output power of 0.5 W, Group 4: Diode laser with output power of 1 W. Thermocouple device was held by experimenter hand and the sensor was in pulp chamber of the teeth. Heat generated during irradiation was reported on LCD screen by NUX Plus software. Repeated measure ANOVA was used in order to compare the temperature before and after laser application. Tukey HSD was used to compare the results between groups. Results Temperature was risen for about 0.54 ± 0.05 °C for group 1, 1.67 ± 0.14 °C for group 2, 4.21 ± 0.83 °C for group 3, and 4. 50 ± 0.32 °C for group 4. The difference between group 1 & 2, 1 & 3 and 1 & 4 was significant (p < 0.0001). Conclusion According to results of this study, Diode (with or without photosensitizer- 0.5 and 1 W) can be safely used as alternative approach for disinfection after caries removal in thermal point of view. [ABSTRACT FROM AUTHOR]

• aPDT using TBO and ICG has no adverse effect on microleakage of class V cavities. • Microleakage in gingival part of cavities reduced significantly after aPDT using ICG. • ICG as photosensitizer is more recommended to be used in aPDT due to its lower degree of microleakage. Considering the importance of disinfecting dentin and enamel after cavity preparation and the possible effect of disinfection methods on induction of various reactions in the tooth structure the aim of the present study was to evaluate microleakage of composite resin restoration after disinfecting the prepared dentin and enamel surface with antimicrobial photodynamic therapy (aPDT) with toluidine blue (TBO) and indocyanine green (ICG). Standard class V cavities were prepared on buccal surface of 71 human premolar teeth. The samples were randomly divided into 3 groups based on disinfection method: Group 1: conventional disinfection method with Phosphoric acid 37% as the control; Group 2: aPDT with TBO and diode laser with wavelength of 635 nm; Group 3: aPDT with ICG and diode laser with wavelength of 808 nm. All the cavities were restored with composite resin (3M™ Filtek™ Z250). After thermocycling and immersing in 0.5% basic fuchsin, the samples were prepared for microleakage evaluation under a stereomicroscope. Data was analyzed with Kruskal-Wallis and Wilcoxon signed-rank tests at P < 0.05. There were no significant differences in the microleakage of occlusal and gingival margins between the TBO and control groups (P > 0.05). Also, the microleackage of occlusal margins between groups was not significantly different (P > 0.05) but microleackage of gingival margins of ICG group was lower than two other groups in a meaningful way (P < 0.05). Photodynamic therapy with ICG as disinfecting agent in cavity preparations before composite resin restorations decreases the microleackage of gingival margins. [ABSTRACT FROM AUTHOR]

Summary: Background: To determine the effect of photoactivated disinfection (PAD) using toluidine blue and a light-emitting diode (LED) in the red spectrum (wave length at 625–635nm) on species associated with periodontitis and peri-implantitis and bacteria within a periodontopathic biofilm. Methods: Sixteen single microbial species including 2 Porphyromonas gingivalis and 2 Aggregatibacter actinomycetemcomitans and a multispecies mixture consisting of 12 species suspended in saline without and with 25% human serum were exposed to PAD. Moreover, single-species biofilms consisting of 2 P. gingivalis and 2 A. actinomycetemcomitans strains and a multi-species biofilm on 24-well-plates, grown on titanium discs and in artificial periodontal pockets were exposed to PAD with and without pretreatment with 0.25% hydrogen peroxide. Changes in the viability were determined by counting the colony forming units (cfu). Results: PAD reduced the cfu counts in saline by 1.42log10 after LED application for 30s and by 1.99log10 after LED application for 60s compared with negative controls (each p <0.001). Serum did not inhibit the efficacy of PAD. PAD reduced statistically significantly (p <0.05) the cfu counts of the P. gingivalis biofilms. The viability of the A. actinomycetemcomitans biofilms and the multi-species biofilms was statistically significantly decreased when PAD was applied after a pretreatment with 0.25% hydrogen peroxide. The biofilm formed in artificial pockets was more sensitive to PAD with and without pretreatment with hydrogen peroxide compared with those formed on titanium discs. Conclusions: PAD using a LED was effective against periodontopathic bacterial species and reduced viability in biofilms but was not able to completely destroy complex biofilms. The use of PAD following pretreatment with hydrogen peroxide resulted in an additional increase in the antimicrobial activity which may represent a new alternative to treat periodontal and peri-implant infections thus warranting further testing in clinical studies. [Copyright &y& Elsevier]

Summary: Background: An important determinant of the clinical applicability and value of antimicrobial photodynamic inactivation (PDI) is the cytotoxicity of the treatment to human cells. We evaluated the in vitro cytotoxicity of PDI to primary human fibroblasts using methylene blue (MB) and toluidine blue O (TBO) as the photosensitizers. Methods: The primary human fibroblasts were exposed to PDI regimes that were used for the inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant Escherichia coli (MDR E. coli). Mitochondrial activity subsequent to exposure was evaluated after 24h using the methylthiazoletetrazolium assay and compared to pretreatment values. Results: Mitochondrial activity of primary human fibroblasts was reduced by 27% after exposure to light (163.8J/cm2) and MB (50μg/ml). At a TBO concentration previously demonstrated to induce 99.91% and 83.2% reduction in a viable count for MRSA and MDR E. coli, respectively, 39.6% of the fibroblasts were photo-inactivated. Conclusion: Our findings showed that MB/TBO-PDI did not induce significant cytotoxic effects on human fibroblasts in culture. [Copyright &y& Elsevier]

Summary: Methylene blue has been widely used since the late 19th century in biomedical research, and was the lead compound in several important clinical areas, including therapeutics for malaria and schizophrenia. The photodynamic therapy (PDT) of cancer and, more recently, of microbial infection (photodynamic antimicrobial chemotherapy (PACT)) has also employed methylene blue and its congeners, among other chemical types, due to the low human toxicities and efficient photosensitising properties of the group. However, little work has been carried out in terms of derivative and structure–activity development, most reports covering standard, commercially available compounds. This review deals with the evolution of phenothiazinium photosensitisers for both PACT and PDT use. [Copyright &y& Elsevier]

• In endodontics, residual intra-canal microorganisms effect the long-term success of the treatment negatively. • In this study, the elimination effectiveness of additional antimicrobial photodynamic therapy on residual microorganisms was tested. • In the present study, all aPDT protocols significantly reduced intra-canal bacterial load compared to initial. • However, there was no difference between MB, TB, and TM-ZnPc in terms of reducing intra-canal bacterial load. Traditional chemomechanical treatment procedures are an indispensable part of endodontic treatment, however, additional treatment approaches such as antimicrobial photodynamic therapy (aPDT) may also be recommended for the elimination of residual microorganisms. In this study, the disinfection efficiency of aPDT performed using methylene blue (MB), toluidine blue (TB), and tetra 2-mercaptopyridine substituted zinc phthalocyanine (TM-ZnPc) was compared in the roots contaminated with Enterococcus faecalis (E. faecalis). Forty-nine teeth with a single root and canal were included in this study. The roots were sterilized, and inoculated with E. faecalis. The roots were kept in an incubator for 30 days to form the biofilm. Forty-five teeth were prepared up to the F3 file of the ProTaperNext system under 2.5 % NaOCL irrigation. The samples were divided into three groups according to the type of used photosensitizer (PS) (n = 15); MB (313 μM), TB (327 μM), and TM-ZnPc (6μM). All PSs were irradiation with a light-emitting diode (LED) lamp (630 nm, 2–4 mW/cm²) for the 60 s. Two microbiological samples of the intracanal content were taken (one before and one immediately after additional aPDT in all groups) using sterile paper points. The colony-forming units per milliliter (CFU/mL) were calculated after 24 h of incubation at 37 °C. After all aPDT protocols, intracanal bacterial load decreased significantly compared to the amount after chemomechanical preparation (P < 0.05). No significant difference in the reduction in intracanal bacterial load was found between the PSs (P < 0.05). In the current study, the aPDT protocol performed with TM-ZnPc provided similar antimicrobial efficacy, although it was used at a lower concentration compared to MB and TB. Therefore, the use of TM-ZnPc in intra-canal disinfection in endodontics seems promising. [ABSTRACT FROM AUTHOR]

Abstract: It is known that Staphylococcus aureus is susceptible to photodynamic inactivation in general, but the significant variation among particular strains in the response to the treatment exists. However, factors that determine the observed phenomenon remain unclear. This study was aimed to explore the PDI effect of two sensitizers (protoporphyrin diarginate and toluidine blue O) against clinical as well as reference strains of S. aureus. Obtained results indicate that the same isolate could be characterized as highly resistant or highly sensitive to PDI according to a sensitizer used. Moreover, the same sensitizing agent could be successfully used for total eradication of some isolates and could be non-effective in the case of other strains. Additionally, changing the photosensitizer, we are able to reverse the PDI “resistant” phenotype into “sensitive” one. Thus, one could conclude that photoinactivation involving several sensitizing agents and several isolates of the same bacterial species should be undertaken to make antimicrobial photodynamic inactivation reliable. [Copyright &y& Elsevier]

• Photodynamic therapy (PDT) is a valid treatment option for treatment of oral lesions. • Toluidine Blue as photosensitizer is still discussed in the treatment of oral lesions. • Leukoplakia is a white patch that can't be identified clinically as any other disease. • This study focuses on feasibility of topical toluidine blue-based PDT for leukoplakia. • Topical toluidine blue PDT is easy to perform, well tolerated and effective method. Photodynamic therapy (PDT) is a minimally invasive method for the treatment of oral leukoplakia (OL) through the activation of a photosensitizer, after exposure to a specific wavelength light source. To investigate the effectiveness of toluidine blue as topical photosensitizer. Eleven patients participated in this study; fifteen oral leukoplakia lesions were treated, in several sessions, with 2.5 % toluidine blue and an LED source of 630 nm wavelength. Patients were evaluated at baseline (t0), at the end of treatment cycles (t1) and one year from the end of treatment (t2). All the treated sites were photographed at each visit. Images were processed with ImageJ 1.52 software in order to obtain the areas (mm2) of the treated lesions. Comparison between data at different follow-up was performed using a paired T-test. At t1, complete response was obtained in six lesions, partial response in seven lesions while only two lesions showed no response. At t2, a further improvement was observed in two patients. The analysis of the areas showed significant reduction of the lesion size from t0 to t1 (p = 0.003), and from t1 to t2 (p = 0.01). Toluidine blue appears to be a promising photosensitizer in the photodynamic therapy of oral leukoplakia. [ABSTRACT FROM AUTHOR]

• TB-PACT combined with GEN significantly inhibited bacterial proliferation. • PACT permeabilized the bacterial membranes and augmented GEN effects. • ROS generation of synergistic combination promoted bacterial inactivation. Bacterial resistance to antibiotics is generally increasing, which has become a great challenge for treating infectious diseases caused by microbes. Photodynamic antibacterial chemotherapy (PACT) has been considered as a promising method for inactivating bacteria. The combination of antimicrobial agent with PACT may provide efficient way against drug-resistant microbe. This study aims to investigate the synergistic effects of PACT mediated by toluidine blue (TB), combined with gentamicin (GEN) on common pathogens Staphylococcus aureus (S. aureus) and multidrug-resistant S. aureus (MDR S. aureus). Alkaline lysis was used to detect the uptake of TB by S. aureus and MDR S. aureus. Plate counting was applied to evaluate the inhibition efficiency of GEN alone, TB-PACT alone, and work together. Flow cytometry and fluorescence microscopy were performed to examine the permeability of bacterial membranes after different treatments. Intracellular and extracellular reactive oxygen species (ROS) were assessed with the assist of H 2 DCF-DA and SOSG probes. TB-PACT combined with GEN led to more pronounced antibacterial effects in S. aureus and MDR S. aureus , as compared with either alone. TB-PACT treatment permeabilized the bacterial membranes, promoted GEN cellular accumulation and augmented the antibacterial efficiency. The intracellular ROS generation by the combination of TB-PACT and GEN was much higher than that of single treatment groups. TB-PACT decreased the GEN cytotoxic threshold and usage, and the synergy of them significantly enhanced the sterilization of S. aureus and MDR S. aureus. [ABSTRACT FROM AUTHOR]