Assessment of sensitivity of selected Candida strains on antimicrobial photodynamic therapy using diode laser 635 nm and toluidine blue - In vitro research.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Comparative Effect of Two Red Lights on Streptococcus mutans Biofilms and Assessment of Temperature Variances in Human Teeth During In Vitro Photodynamic Antimicrobial Chemotherapy.

Objective: The goals of this investigation were to compare the effect of photodynamic antimicrobial chemotherapy (PACT) with two different red lights on in vitro Streptococcus mutans biofilms, as well as to assess the temperature variances caused by PACT on human teeth. Methods: S. mutans biofilms (n = 3) were grown on hydroxyapatite disks, and the antimicrobial effect of PACT was evaluated using toluidine blue O (100 µg/mL) associated with Laserbeam® (LB 56.6 J/cm2) and LumaCare™ (LC -56.6, 158.5, 317.0, and 475.6 J/cm2). Pulpal temperature variances were analyzed using a digital thermocouple placed into the pulp chamber and positioned at the cement-enamel junction level of five teeth samples during irradiation times of 300, 600, and 900 sec for LB, and 22, 60, 120, and 180 sec for LC. The mean average temperature variance was calculated for each group. All data were analyzed through analysis of variance. Results: LB (900 sec) and LC (22 sec) induced similar reductions in the viability of microorganisms. LB did not cause statistically significant increase of temperature, regardless of experimental time, and LC caused temperature increase within the safe spectrum up to 60 sec. Conclusions: PACT seems to be a minimal invasive approach for reducing the viability of cariogenic bacteria. Thus, when applied in vitro for times equal or inferior to 900 and 60 sec for LB and LC, respectively, these light sources might be considered harmless to tooth structures.

The effect of sublethal photodynamic therapy on the expression of Enterococcal surface protein (esp) encoding gene in Enterococcus faecalis: Quantitative real-time PCR assessment.

BACKGROUND: During antimicrobial photodynamic therapy (aPDT) in the treatment of endodontic intracanal infection, it is extremely likely that microorganisms would be exposed to sub-lethal doses of PDT (sPDT). Although sPDT would not result in microorganism death, it can considerably influence microbial virulence. This study evaluated the effect of sPDT on gene expression of Enterococci surface protein (esp) as a major virulence factor for biofilm formation in Enterococcus faecalis. MATERIALS AND METHODS: The lethal and sub-lethal potential of aPDT using indocyanine green (ICG), toluidine blue O (TBO), and methylene blue (MB), as the photosensitizers (PSs) and 660, 635, and 810 nm diode laser against E. faecalis was analyzed using colony-forming unit assays. Considering sub-lethal doses of PSs and photo-irradiation time of the diode laser, the expression of esp was evaluated through quantitative Real-time PCR (qRT-PCR). RESULTS: ICG at a concentration of 31.2-1000 µg/mL and both TBO and MB at 6.2-100 µg/mL significantly reducedE. faecalis growth. 660, 635, and 810 nm diode laser with energy density of 93.75-140.62, 137.5-206.25, and 31.25-62.5 J/cm2, reduced the bacterial count. ICG with a concentration of 15.6 µg/mL and irradiation time of 0.5 min, TBO with a concentration of 3.1 µg/mL and irradiation time of 3 min, and MB with a concentration of 3.1 µg/mL and irradiation time of 3 min were found as a sPDT dose against E. faecalis. The esp expression was significantly reduced at sub-lethal doses of ICG compared to TBO (2 fold) and MB (2.4 fold). CONCLUSION: Although all tested PSs showed bacterial reduction, ICG may be considered as the best PS in treating endodontic infection due to its higher efficacy in reduction of esp expression which has a main role in biofilm formation of E. faecalis.

Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment.

BACKGROUND: We have previously suggested that the Toluidine Blue (TB) test can be used for sperm chromatin structure assessment. In this study, we wished to evaluate the clinical applicability of the TB test in assessing male fertility potential using well-defined groups of fertile and infertile men. METHODS: Sixty-three fertile and 79 infertile men were tested. Infertility thresholds for the proportion of sperm with abnormal [TB dark cells (TBDCs)] and normal [TB light cells (TBLCs)] chromatin structure were set by the ROC curve analysis. RESULTS: Thresholds of 45% TBDC and 20% TBLC were highly predictive for infertility (specificity of the test: 92 and 90%, respectively), but they were poor predictors of the fertility (sensitivity of the test: 42 and 32%, respectively). Odds ratio for infertility was 7.5 [95% confidence interval (CI): 2.7-20.8] when the 45% TBDC threshold was used and 4.4 (95% CI: 1.7-11.6) when the 20% TBLC threshold was used. CONCLUSIONS: The TB test can be suggested for clinical use as a complementary test for standard semen analysis to diagnose male infertility.

An assessment of the activity of a novel light-activated antimicrobial coating in a clinical environment.

Cellulose acetate coatings containing the light-activated antimicrobial agents toluidine blue O and rose bengal have previously been shown to be successful in killing a range of microorganisms. Here, we report on the ability of these coatings to achieve reductions in the microbial load on surfaces in a clinical environment.

Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence.

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.