Photodynamic therapy against multidrug-resistant oral strains of Candida spp
Introduction. The advent of Candida ssp. strains that are resistant to different drugs, or MDR strains, highlights the necessity of creating novel antimycotics that are both effective and safe for systemic health. Within this framework, photodynamic therapy (PDT) may be a viable option for treating yeast infections. In this context, the purpose of this work is to evaluate and compare, through a clinical-laboratory multidisciplinary approach, commercially available protocols and new procedures based on new photosensitizers and/or different light radiation sources. In fact, this thesis is aimed at three main objectives: (i) First, describe and compare the clinical effectiveness of the main PDT therapies already used against Candida infection; (ii) conduct a strict investigation of new PDT procedures by using natural compounds as photosensitizers (PT); and (iii) fine-tune new laboratory procedures to evaluate the infection in infected and then PDT-treated cell culture. Methods. A set of in vitro and in vivo studies were performed. The in vitro studies were executed with oral MDR isolates of C. albicans, C. krusei, and C. glabrata, these fungal strains were used from cell suspension in culture liquid medium at their log growth phase. The laboratory procedures were addressed according to EUCAST protocols, which were modified to conform to the PDT treatment, and they could be divided into three main procedures: (i) evaluation with the agar diffusion susceptibility test by Kirby Bauer; (ii) growth curve inhibition and antibiofilm activity through the microplate dilution method; (iii) activity of PDT against a subprosthetic in vitro C. glabrata infection. For these in vitro procedures, a large set of commercial and natural PT as well as different light sources were evaluated. The antimycotic activity were in fact evaluated by using these subsequent commercials formulates: Curcumin/H2O2, Toluidine Blue 1% and Methylene Blue 1%. The natural compounds evaluated as PT were: olive oil, Lactoferrin, Saffron, Pompia a Strawberry tree juices. These extracts were potentiated by using 3% H2O2 and activated with different lights. Lab procedures to evaluate the prognosis in infected and then PDT-treated patients. The use of MiRNAs 146a and 155 as a predictive targets of infection level and the effectiveness of PT was assessed in a first-line in vitro experiment by using HEK cells infected with an MDR strain of C. albicans. From culture medium, at different times, total RNA was extracted, and MiRNAs were quantified by real-time qPCR protocol. In vivo clinical study. A set of 12 patients with refractory Candidiasis have been treated with commercial PDT protocols, thus a clinical follow up was performed to evaluate the healing by a clinical and laboratory evidences.Results.Among the commercial-based PDT protocols, the in vitro experiments have suggested the main role of curcumin-based treatment; in fact, in comparison to Toluidine Blue 1% and Methylene Blue 1%, the inhibitions halos were 46 and 0 mm, respectively. In the prosthetic device infected with C. glabrata, the curcumin/H2O2 PDT, decreased the CFU values from 1500/cm2 to 20 CFU/cm2 after the PDT treatment. By using natural PSs, good activity was observed with olive oil, lactoferrin, and saffron. In the cell culture model, a variation in the expression patterns of 146a and 155 was observed within 2 minutes of infection. Clinical evaluation: on 12 treated patients, in 9 cases, we had clinically complete healing; in three cases, we had partial results.Conclusions. Experimental results suggest the importance of photodynamic therapy in the treatment of chronic candidiasis in the mouth. This study also highlights the possibility of using new natural photosensitizers as natural, non-toxic products. Furthermore, MiRNA's evaluation as a predictive laboratory analysis for Candida infection could be an efficient tool for controlling the infection progression before and after PDT.
Settore MED/50 - Scienze Tecniche Mediche Applicate
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