The importance of combining methods to assess Candida albicans biofilms following photodynamic inactivation.

BACKGROUND: Methylene blue (MB)-mediated photodynamic inactivation (PDI) has shown good results in killing Candida spp. Although MB solutions are commonly used, new formulations have been designed to improve PDI. However, chemical substances in the formulation may interfere with the PDI outcome. In this sense, different methodologies should be used to evaluate PDI in vitro. Herein, we report different methodologies to evaluate the effects of PDI with an oral formulation (OF) containing 0.005% MB on Candida albicans biofilm. METHODS: Biofilms were treated using the MB-OF, with 5 min pre-irradiation time and exposure to a 640 nm LED device (4.7 J/cm2). PDI was evaluated by the XTT reduction test, counting the colony forming units (CFU), a filamentation assay, crystal violet (CV) staining, and scanning electronic microscopy (SEM). RESULTS: PDI was able to reduce around 1.5 log10 CFU/mL, even though no significant differences were noted in metabolic activity in comparison to the control immediately after PDI. A significant decrease in yeast to hyphae transition was observed after PDI, while the biofilm exhibited flattened cells and a reduced number of yeasts in SEM. The CV assay showed increased biomass. CONCLUSION: MB-OF-mediated PDI was effective in C. albicans biofilms, as it significantly reduced the CFU/mL and the virulence of surviving cells. The CV data were inconclusive, since the OF components interacted with the CV, making the data useless. Taken together, our data suggest that the association of different methods allows complementary responses to assess how PDI mediated by a formulation impacts biofilms.

Controlling methylene blue aggregation: a more efficient alternative to treat Candida albicans infections using photodynamic therapy.

Methylene Blue (MB) has been widely used in antimicrobial Photodynamic Therapy (aPDT), however, the mechanisms of action (Type I or Type II) are defined by its state of aggregation. In this sense, the identification of the relationships between aggregation, the mechanisms of action and the effectiveness against microorganisms, as well as the establishment of the means and the formulations that may favor the most effective mechanisms, are essential. Thus, the objective of this study was to assess the in vitro aPDT efficacies against Candida albicans, by using MB in vehicles which may influence the aggregation and present an oral formulation (OF) containing MB, to be used in clinical aPDT procedures. The efficacy of MB at 20 mg L-1 was tested in a range of vehicles (water, physiological solution - NaCl 0.9%, phosphate saline buffer - PBS, sodium dodecyl sulfate 0.25% - SDS and urea 1 mol L-1) in a C. albicans planktonic culture, when using 4.68 J cm-2 of 640 ± 12 nm LED for the irradiations, as well as 5 minutes of pre-irradiation time, together with measuring the UFC mL-1. Based upon these analyses, an OF containing MB in the most effective vehicle was tested in the biofilms, as a proposal for clinical applications. When comparing some of the vehicles, sodium dodecyl sulfate was the only one that enhanced an MB aPDT efficacy in a planktonic C. albicans culture. This OF was tested in the biofilms and 50 mg L-1 MB was necessary, in order to achieve some reduction in the cell viabilities after the various treatments. The light dosimetries still need further adaptations, in order for this formulation to be used in clinical applications. The present research has indicated that the development of this formulation for the control of MB aggregations may result in more effective clinical protocols.

Oral hygiene in intensive care unit patients with photodynamic therapy: study protocol for randomised controlled trial.

BACKGROUND: In intensive care units (ICUs), nosocomial infections are prevalent conditions and they have been related to high mortality indexes. Some studies have suggested that inefficient oral hygiene and ventilator-associated pneumonia (VAP) are related. Nowadays, in the Brazilian public health system there is no well-defined protocol for oral hygiene in an ICU. Due to the drawbacks of the use of antibiotics, photodynamic therapy (PDT) has emerged as an interesting technique in order to reduce antimicrobial-resistant pathogens. Methylene blue (MB) is the most common chemical agent for PDT in Brazil. However, new formulations for improved effectiveness are still lacking. The objective of this study is to evaluate the use of an MB mouthwash as an effective oral-hygiene procedure in an ICU and to show that oral hygiene using PDT with MB mouthwash may reduce VAP frequency to rates similar to, or higher than, chlorhexidine. METHODS: Phase 1 will evaluate the most effective cleaning procedure, while phase 2 will correlate oral hygiene to VAP incidence. At the start of phase 1, the ICU patients will be randomly allocated into three different groups (10 patients/group): the efficacy of chlorhexidine, classical MB-PDT, and mouthwash MB-PDT will all be measured for the quantification of viable bacteria, both pre- and post-treatment, by a Reverse Transcription Polymerase Chain Reaction (RT-PCR). In phase 2, the most effective procedure found in phase 1 and a mechanical cleaning with filtered water will be carried out daily, once a day, over 5 days, with a total of 52 ICU patients randomly allocated into the two groups. The clinical records will be evaluated in order to find any pneumonic diagnoses. DISCUSSION: Since a variety of bacterial species are related to VAP, a universal primer for bacteria will be used in order to quantify the total bacteria count in the participants' samples. In order to quantify only the living bacteria before DNA extraction, the samples will be treated with propidium monoazide. This will infiltrate the dead bacteria and will intercalate the DNA bases, avoiding their DNA amplification. This will be the first trial to evaluate MB-PDT in a mouthwash formula that can increase the effectiveness due to the control of MB aggregation. The results of this study will be able to generate an easy and low-cost protocol to be used in an ICU for the Brazilian public health system. TRIAL REGISTRATION: This protocol was approved by the Research Ethics Committee of the Conjunto Hospitalar do Mandaqui (1.317.834, CAAE: 49273515.9.3001.5551) and it was registered in Registro Brasileiro de Ensaios Clínicos (ReBEC number: RBR-94bvrc;). First received: 12 July 2015; 1st version 6 June 2016. Data will be published in a peer-reviewed journal.