## 3 Results and Discussion  
The biocidal effect on _E. faecalis_ of three different protocols was evaluated by qPCR reactions. In the qPCR tests, an _E. faecalis_ concentration value of \(5.7\times 10^{5}\) was identified before applying any type of treatment. The mechanical treatment reduced the _E. faecalis_ concentration to \(1.02\times 10^{5}\). The mechanical and O\({}_{3}\) aq treatment induced an additional reduction to \(4.87\times 10^{4}\). The complete treatment protocol (mechanical + ozonated water + laser) led to no _E. faecalis_ being identified in the endodontic space (Figure 5). The diagnosis for the MRFM was established by considering the patient symptoms and clinical signs and by using additional X-ray investigations that showed the well-defined area of radiolucency, with the disappearance of the lamina dura and periodontal ligaments, associated with the negative response to pulpal vitality tests. Clinical evaluation of the patient two weeks after the endodontic treatment showed no clinical symptomatology, with negative percussion and palpation, and the visual intraoral evaluation showed healing of the fistula tract. The radiographic evaluation after two months indicated that the pathological apical radiolucency was reduced to a minimum. The number of therapy sessions needed for complete decontamination of the root canals is still among controversial debate topics in the endodontic literature. In this study we chose to make the treatment and microbiological analysis in a single visit. This decision relies on the results of Cochrane systemic analysis conducted in 2008, which showed that there is no significant difference in treatment efficiency according to the number of dental visits [29]. Supplementary, in our in vivo study the conventional chemical disinfectants were completely replaced by ozone and antimicrobial photodynamic therapy. Our data obtained by associating these two methods in root canal decontamination (laser radiation and ozone water) represent a novelty in the research field. [PERSON] et al. in 2008 showed that O\({}_{3}\) aq used as an irrigation agent significantly reduces the number of _Candida albicans_[30] and _E. faecalis_ in human root canals [31, 32]. [PERSON] et al. in 2007 scientifically proved that ozone has anti-inflammatory and immunomodulatory properties, exerting inhibitory effects on the NF-kB system, which is crucially implicated in the inflammatory, immune and apoptosis processes [33]. Due to these results, the use of ozone in dentistry has become more frequent. In endodontics the use of ozone relies on its antimicrobial efficacy, as well as on its oxidation capacity to reduce the number of microorganisms in the root canal [34, 35, 36]. Nevertheless, the conclusion of a recent systematic review was that ozone therapy should not replace or complement the use of NaCl, revealing that the performance in microbial load reduction of ozone is inferior to that of NaOCl [37]. Instead, the laser interacts with water molecules (by irradiation) and produces a cavitation phenomenon that allows better intracanalar penetration of the irrigation solution [38, 39]. One of the most important actions performed by the dental laser is the heating action, but this can also be considered as a disadvantage. A 10 \({}^{\circ}\)C rise in temperature for 1 min can irreversibly damage the  
Figure 5: qPCR test values of _E. faecalis_ for the four protocol stages. periodontal tissues. The heating effect can be reduced by optimizing the application process (time and intensity). Special attention was paid to all operating components and stages of the proposed experimental therapeutic protocol, which already were optimized in previous studies [1; 35]. Thus, in our study diode laser irradiation was performed at C/P-Mode-2 W (four times) for 15 s, and each irradiation was followed by 20 s of O\({}_{3}\) aq irrigation.