|Year : 2010 | Volume
| Issue : 3 | Page : 341-348
|The use of ozonated water and 0.2% chlorhexidine in the treatment of periodontitis patients: A clinical and microbiologic study
Durga Kshitish, Vandana K Laxman
Department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India
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|Date of Submission||01-Oct-2009|
|Date of Decision||12-Nov-2009|
|Date of Acceptance||21-May-2010|
|Date of Web Publication||29-Sep-2010|
| Abstract|| |
Background: The development of periodontal disease has been thought to be associated with several restricted members of the oral anaerobic species, such as black-pigmented Porphyromonas species and Actinobacillus actinomycetemcomitans (Aa), in the subgingival environment. Apart from bacteria, certain viruses and fungi that are associated with periodontal disease are also present in the subgingival plaque .
Materials and Methods: A randomized, double-blind, crossover split-mouth design was performed. A total of 16 patients suffering from generalized chronic periodontitis were selected for the study. The study period of 18 days was divided into two time-intervals, i.e. baseline (0 days) to 7 th day, with a washout period of 4 days followed by a second time interval of 7 days. The use of ozone and chlorhexidine gluconate (CHX) irrigation was randomized. Both the patient and the clinician evaluating the clinical parameters were blinded regarding the type of irrigation used.
Results: The interpretation of clinical and microbial data is from baseline to 7 th day. A higher percentage of plaque index (12%), gingival index (29%) and bleeding index (26%) reduction was observed using ozone irrigation as compared to chlorhexidine. The percentile reduction of Aa (25%) using ozone was appreciable as compared to no change in Aa occurrence using chlorhexidine. By using O 3 and chlorhexidine, there was no antibacterial effect on Porphyromonas gingivalis (Pg) and Tannerella forsythensis. The antifungal effect of ozone from baseline (37%) to 7 th day (12.5%) was pronounced during the study period, unlike CHX, which did not demonstrate any antifungal effect.
Conclusion: Ozone may be considered as an alternative management strategy due to its powerful ability to inactivate microorganisms. Also, there is growing evidence that ozone can be employed as a useful therapeutic agent in both dentistry and medicine.
Keywords: Bacteria, chlorhexidine, fungus, ozone, single irrigation, viruses
|How to cite this article:|
Kshitish D, Laxman VK. The use of ozonated water and 0.2% chlorhexidine in the treatment of periodontitis patients: A clinical and microbiologic study. Indian J Dent Res 2010;21:341-8
The development of periodontal disease has been thought to be associated with several restricted members of oral anaerobic species, such as black-pigmented Porphyromonas species and Actinobacillus actinomycetemcomitans itans (Aa), in the subgingival environment. Apart from bacteria, certain viruses and fungi are also present in the subgingival plaque that have been associated with periodontal disease. The involvement of Herpes viruses in the etiology of periodontal diseases was suggested by their presence in the gingival tissue, gingival crevicular fluid and subgingival plaque, in the presence of periodontal disease.  Candida albicans has been recovered from periodontal pockets in a large number of patients with chronic periodontitis and had been found to be invading the gingival connective tissue in patients with juvenile periodontitis. 
|How to cite this URL:|
Kshitish D, Laxman VK. The use of ozonated water and 0.2% chlorhexidine in the treatment of periodontitis patients: A clinical and microbiologic study. Indian J Dent Res [serial online] 2010 [cited 2019 Sep 17];21:341-8. Available from: http://www.ijdr.in/text.asp?2010/21/3/341/70796
Removal of dental plaque thus forms an important part of controlling and treating periodontal disease, which brings about both qualitative as well as quantitative changes in the subgingival microflora. A number of chemical adjuncts have been used to improve the outcome of mechanical oral hygiene procedures, one of which is chlorhexidine, a broad-spectrum antiseptic with pronounced antimicrobial effects on Gram-positive as well as Gram-negative bacteria, some viruses and fungi. ,
One inference of the ecological plaque hypothesis is that disease might be prevented not only by inhibiting the putative pathogens but also by interfering with the factors responsible for the transition of the plaque microflora from the commensal to a pathogenic relationship with the host.  The early studies confirm the theoretical basis of the ecological plaque hypothesis by showing that a preventing strategy that interferes with critical event in the breakdown of microbial homeostasis in plaque can shift the ecological balance of plaque back toward one that is compatible with dental health. 
A consideration of the principles behind the ecologic plaque hypothesis can lead to the identification of new strategies to prevent disease and offer new perspectives on existing approaches. Thus, the preventing strategies based on the ecological plaque hypothesis include altering the subgingival environment by various methods, which also includes the application of oxygenating and redox agents. 
An alternative approach to conventional antimicrobial or antiseptic agents in the suppression of subgingival bacteria is to inhibit their growth by changing the subgingival environment, which has been shown to be highly anaerobic with a prevailing low oxygen tension.  First advocated by Dunlop in 1913, various agents such as molecular oxygen,  hyperbaric oxygenation  and hydrogen peroxide have been applied.  It has been shown that repeated subgingival oxygen irrigation in previously untreated deep periodontal pockets resulted in a significant clinical improvement of the periodontal baseline conditions.  Recently, ozone therapy is gaining popularity in various treatment modalities in the field of medicine, dentistry, veterinary, food industry, water treatment, etc. In dentistry, ozone is being successfully utilized for the treatment of dental caries.
Although ozone has been shown to be detrimental against bacteria, viruses, fungi and protozoa in vitro, less attention has been paid to the antibacterial, antiviral and antifungal properties of ozonated water on periodontopathic microorganisms in vivo. Currently, the established oral antiseptics for periodontal treatment include chlorhexidine gluconate (CHX, 0.2-2%). Regarding side-effects, it is known that chlorhexidine may cause mucosal desquamation, impaired wound healing and fibroblast attachment to the tooth surfaces, tooth staining and altered taste sensation. In proposing ozone as another potential antimicrobial for use in the oral cavity, it is important to compare its potential with that of the potential of established agents . Ozone is currently being discussed in dentistry as a possible alternative antiseptic agent. Its high antimicrobial power without the development of drug resistance has been noted in water purification and food preservation. Recent investigations have reported antimicrobial effects on oral pathogens of both gaseous and aqueous forms of ozone, and the effectiveness of ozone in the treatment of oral diseases is currently a subject of intense research. 
The MEDLINE search revealed lack of periodontal literature on the effects of ozone on clinical parameters of periodontal inflammation. In vivo antibacterial, antiviral and antifungal effect of ozone has been attempted for the first time in this preliminary clinical trial. Hence, the aim of the present study was to evaluate the effect of oral irrigation with ozonated water and 0.2% chlorhexidine in moderately deep periodontal pockets on the clinical parameters and microbial profile in subgingival plaque of chronic and aggressive periodontitis.
Hence, the objectives of the study include evaluation and comparison of the effects of oral irrigation with ozonated water and 0.2% chlorhexidine on clinical parameters such as Plaque Index, Gingival Index and Gingival Bleeding Index. A second objective of the study was to assess and compare the effects of oral irrigation with ozonated water and 0.2% chlorhexidine on microorganisms such as bacteria, including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis (Tf), viruses such as Herpes simplex virus (HSV-1 and 2), Human Cytomegalo-virus (HCMV) and Epstein Baar virus (EBV), and the fungus, Candida albicans.
| Materials and Methods|| |
The subjects for this study were selected from the Out-Patient Department of Periodontics, College of Dental Sciences, Davangere, Karnataka. Patients of both sexes within the age limit of 20-60 years were included in the study.
A randomized, double-blind, crossover split-mouth design was performed. A total of sixteen patients suffering from generalized chronic and aggressive periodontitis were selected for the study. Patients who were pregnant or lactating; suffering from any known systemic diseases, who had received any surgical or non-surgical therapy 6 months prior to the start of the study, who had received any antibiotic therapy in the last 6 months, who had received any chemotherapeutic mouth rinses and oral irrigation during the past 6 months and who were smokers were excluded from the study. Ethical clearance was obtained from the ethical committee of the College of Dental Sciences, Davangere, India. Data collection was performed between August 2006 and October 2006. All participants gave informed consent.
The study period of 18 days was divided into two time-intervals, i.e. baseline (0days) to 7 th day, with a washout period of 4 days followed by second time interval of 7 days. The use of ozone and CHX irrigation was randomized. Both the patient and the clinician evaluating the clinical parameters were blinded regarding the type of irrigation used.
A split-mouth design was used in this study for subgingival irrigation of each half of the mouth with either ozone or chlorhexidine at different time intervals. At baseline and on the 7 th day, the clinical parameters, viz. Plaque Index (Silness and Loe, 1964), Gingival Index (Loe and Silness, 1963) and Gingival Bleeding Index (Ainamo & Bay, 1975), were recorded and the subgingival pooled plaque sample was collected from the selected sites. Later, one-half of the mouth (upper and lower quadrants) was irrigated either with 2% CHX solution via a magnetostrictive ultrasonic insert with a medium-power setting (Dentsply cavitron: DENTSPLY Tulsa Dental Specialties, DENTSPLY (International), 5100 E. Skelly Dr. Ste (300) Tulsa, OK 74135, USA ) or ozonated water was delivered using a modified needle attached to an irrigation device, "Kent ozone dental jet TY-820" (Kent Dental Care Products, USA). After a washout period of 4 days, the clinical parameters were re-assessed and the pooled plaque samples were collected from the irrigation sites. The contralateral side of the mouth was subjected to irrigation in the second interval. The procedure of clinical assessment and collection of pooled plaque sample were repeated on the 18 th day.
The patients were subjected to oral irrigation with ozonated water that was released from an irrigation device, "Kent ozone Dental Jet TY-820" (Kent Dental Care Products), to the selected one-half of the mouth.
The device released a single pulsating stream of ozonated water from the nozzle, which could be adjusted for different speeds and pressures ranging from 350 to 500 kPa (kilo pascals) and an ozone output of 0.082 mg/h, at a noise output of <70 dB (decibels) and water outflow of ≥450 ml. To facilitate subgingival ozone irrigation, a 20-gauge blunt needle was bent and attached to the tip of the nozzle of the ozone dental jet holder. The needle was inserted 3 mm subgingivally and irrigated with ozonated water. A total time of 5-10 min was spent for irrigation of the split-mouth sites.
The sites on the other half of the mouth were irrigated with a commercially available 0.2% CHX solution (ORinse; Centaur Pharmaceuticals Pvt. Ltd., Mumbai, India) delivered via a magnetostrictive ultrasonic insert (Dentsply Cavitron 25K FSI-1000-62: DENTSPLY Tulsa Dental Specialties, DENTSPLY (International), 5100 E. Skelly Dr. Ste (300), Tulsa, OK 74135, USA ) with a medium power setting. A total time of 5-10 min was spent for irrigation of the split-mouth sites.
After irrigation, the patients were instructed to perform regular oral hygiene habits, i.e. twice-daily brushing by "roll-on technique" for a minimum of 2 min, using a standard tooth brush (Colgate Super Flexible with medium consistency bristles) and tooth paste (Colgate dental cream) provided to them. The patients were dispersed and instructed to report on the subsequent 7 th day.
The pooled plaque samples collected in Eppendorf vials containing transport media, viz. TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) and Thioglycolate broth were sent to the microbiological laboratory within 24 h of collection, where they were subjected to multiplex polymerase chain reaction (PCR) analysis and culture tests. The following bacteria and viruses were analyzed by PCR analysis:
Bacteria - Aggregatibacter actinomycetemcomitans (Aa)
Porphyromonas gingivalis (Pg)
Tannerella forsythensis (Tf)
Viruses - Herpes simplex virus (HSV-1 and 2)
Epstein Barr virus (EBV)
Human cytomegalovirus (HCMV)
Fungus - Candida albicans was analyzed by culture technique using Sabouraud's agar medium
Processing of samples
The samples, on receipt in the laboratory, were stored at -20°C until DNA extraction and multiplex PCR were performed.
- The specimen was centrifuged at 10000 rpm in the Eppendorf vial.
- The deposit was washed at least three times with 0.5 ml of TE buffer. Washing was performed by centrifugation at 10,000 rpm for 5 min each time and discarding the supernatant.
- The deposit was treated with 0.5 ml of lysis buffer 1 (Tris-EDTA and 0.5% Triton-X 100 as lysis agent) for 5 min after proper mixing.
- The sample was further centrifuged at 10,000 rpm, the supernatant was discarded and 50 μl of Lysis buffer 2 and freshly prepared Proteinase K solution with a final concentration of 100 μg/ml were also added.
- The tube was kept at 65°C for 2 h in a water bath for Proteinase K to act on and digest the protein.
- After this, the sample was exposed to 95°C for 10 min to destroy the Proteinase K.
- Extracted DNA was kept at -20°C until further processing by PCR.
The following oligonucleotide primers were used from 5 ' to 3 ' :
H 1 P 32 with a sequence, TGGGACCATGCCTTCTTGG at primer position 6621-6640.
H1M32 with a sequence, ACCCTTAGT CAGACTCTGTTACTTACCC at primer position 6741-6768.
H2M40 with a sequence, GTACAGACCTTCGGAGG at primer position 56320-56336.
H2P4 with a sequence, CGCTTCATCATGGGC at primer position 56533-56547.
CP 15 with a sequence, GTACACGCACGCTGGTTACC at primer position 8868-8887.
CM3 with a sequence, GTAGAAAGCCTCGACATCGC at primer position 9105-9124.
EP5 with a sequence, AACATTGGCAGCAGGTAAGC at primer position 775-794.
EM3 with a sequence, ACTTACCAAGTGTCCATAGGAGC at primer position 935-957.
With a sequence, CAGCAAGCTGCACAGTTTGCAAA.
With a sequence, AGGCAGCTTGCCATAATGCG and ACTGTTAGCAACTACCGATGT.
With a sequence, TACAGGGGAATAAAATGAGATACG and ACGTCATCCCCACCTTCCTC.
DNA amplification using multiplex PCR
To optimize the multiplex PCR, a series of titrations of primer concentrations and deoxynucleotide triphosphate (dNTP) levels were performed.
Primer concentrations of 10, 25, 50 and 100 pmol from each primer pair were titrated simultaneously with dNTP (0.1, 0.2 and 0.3 mM concentrations of each of the dNTPs.).
The amplification was carried out in 40 cycles of 30 s at 94°C, 40 s at 60°C and 50 s at 72°C in a 50-μl final volume containing 5 μl of 10x reaction buffer (Stratagene, La Jolla, CA, USA), 0.2 mM concentrations of each dNTP, 10 pmol of each of the seven primers and 2.5 U of cloned Pfu DNA polymerase (Stratagene). Five microliters of the appropriate DNA sample was added to the reaction mixture. After the last cycle, the samples were incubated for 15 min at 78°C to complete the extension of primers.
After DNA amplification by PCR, the visualization and analysis of the PCR products was carried out by 2% agarose in Tris-acetate EDTA buffer with 5 μg/ml of ethidium bromide. The samples were subjected to electrophoresis for a minimum of 2 h at a current of 16 volts/gel, with each batch molecular weight standard also run for comparison along with it. After this the gel was exposed to a UV light transilluminator (DNR Bioimaging Systems: Biocompare, 395 Oyster Point Blvd., #321, South San Francisco, CA 94080, United States of America) in a dark room and the image was captured with a digital camera for further analysis. The various microorganisms were identified based on the formation of bands in the form of ladders in the captured image.
Culture technique for Candida albicans
The pooled plaque samples that was in vials containing thioglycolate broth were transported to Sabouraud's agar medium.
The samples were incubated in Sabouraud's medium at 37°C for 48 h and were observed for colony growth. The medium was then stained using Gram's stain and the yeast cells were identified. Large Gram-positive oval to circular cells are diagnostic of yeast cells.
Confirmation test (Germ tube method): Saline and human serum was inoculated on the colonies for a few hours. Presence of Candida albicans could be visualized in a light microscope as hyphae without constriction at their bases.
Results were expressed as mean±SD and proportions as percentages. Intragroup comparisons were made by paired t-test and unpaired t-test for intergroup comparisons. Categorical data were analyzed by Fisher's exact test.
For all the tests, a P-value of 0.05 or less was considered for statistical significance.
| Results|| |
The results of the study are reported in [Table 1],[Table 2],[Table 3],[Table 4],[Table 5],[Table 6]. The interpretation of clinical and microbial data is from baseline to 7 th day. The higher percentage of Plaque Index (12%), Gingival Index (29%) and Bleeding Index (26%) reduction was observed using ozone irrigation as compared to chlorhexidine. The percentile reduction of Aa (25%) using ozone was appreciable as compared to no change in Aa occurrence using chlorhexidine. By using O 3 and chlorhexidine, there was no antibacterial effect on Pg and Tf. The antifungal effect of ozone from baseline (37%) to 7 th day (12.5%) was pronounced during the study period, unlike CHX, which did not demonstrate any antifungal effect. The antiviral effect of ozone was not seen for HSV-1, HCMV and EBV. Chlorhexidine proved different by exhibiting an antiviral effect against HSV-1 and HCMV. The HSV-2 was not found in any of the subgingival plaque samples.
|Table 3 :Comparison of mean values of Gingival Bleeding Index (% of sites) |
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|Table 4 :Comparison of occurrence of various bacteria at different time intervals |
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|Table 5 :Comparison of occurrence of fungus at different time intervals |
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|Table 6 :Comparison of occurrence of viruses at different time intervals |
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The percentage of occurrence of Pg, Tf and HSV-1 was higher on the fourth day, which returned to baseline value on the 7 th day, using ozone irrigation. In the chlorhexidine group, the percentage of Pg was higher, which, however reached baseline levels on the 7 th day, but the percentages of Tf and EBV were lower on the 4 th day and reached the baseline levels.
| Discussion|| |
In the 1998 European Workshop on mechanical plaque control, a policy statement was adopted that states, "Forty years of experimental research, clinical trials and demonstration projects in different geographical and social settings have confirmed that effective removal of dental plaque is essential to dental and periodontal health throughout life." Therefore, the principal objective of periodontal therapy is to eliminate the subgingival bacterial presence and, by subsequent supragingival plaque control measures, prevent or minimize recolonization of the subgingival area by pathogenic microflora. Elimination of the subgingival microflora may be achieved mechanically. The mechanical methods of plaque removal can also be supplemented by antimicrobial agents.
The most common method for controlling the growth of dental plaque is mechanical. However, the effectiveness of this method is limited by such factors as individual motivation, inaccessibility to periodontal pockets, concave tooth surfaces and the margins of restorations. Alternatives to mechanical plaque control are therefore being sought. Chlorhexidine has emerged as an important oral antibacterial agent and adjunct to periodontal therapy. It is a broad-spectrum antiseptic with pronounced antimicrobial effects on Gram-positive as well as Gram-negative bacteria, some viruses and fungi. ,
An extensive review of the English literature has not revealed previous attempts to control the formation and development of plaque in vivo by delivery of gases to the region. Gases may be more advantageous for use intraorally than liquids because of their different physical characteristics and diffusion potentials. Socransky reviewed the possible pathogenic role of anaerobic bacteria isolated from dental plaques associated with periodontal lesions. Oxygen has been used in the past for delivery into the periodontal pockets as few reports have linked the presence of obligate anaerobic organisms in the dental plaque. But, it was shown that direct oxygen gas application has no effect on plaque formation.  In contrast, another experiment conducted to assess the effect of repeated subgingival oxygen irrigations in previously untreated periodontal pockets has shown a significant clinical improvement of the periodontal baseline conditions. 
Apart from direct oxygen irrigation, a few oxygenating agents such as sodium monohydrate, sodium monoxychlorosene, carbamide peroxide and sodium borate peroxyhydrate were applied to areas of wounded rat oral tissue. All of these showed complete healing in a shorter time than normally required.  Recently, an allotropic form of oxygen, ozone, is being used in dentistry for the treatment of dental caries. Ozone gas application for a period of 10 s was capable of reducing the numbers of microorganisms associated with primary root carious lesions in vitro.  Four in vitro studies have reported the antimicrobial effect of ozonated water. Ozonated water has been shown to be effective against Candida albicans, adhering to acrylic denture plates,  on Enterococcus faecalis  and Streptococcus mutans nal tubules  and periodontopathic bacteria such as Aa and Pg in vitro.  Ozonated water has also been shown to be effective on root surfaces after extraoral rinsing for decontamination of avulsed teeth in vitro. 
Although there are studies related to the use of ozonated water on oral microorganisms in vitro, no literature exists till date on the in vivo effect of ozonated water on oral and, in particular, periodontopathic microorganisms. Ozone is a selective oxidant and effects only certain compounds, but when it dissolves in water, it becomes highly unstable and rapidly decomposes through a complex series of chain reactions. , As a result, hydroxyl (OH) radicals are generated, which are among the most reactive oxidizing species. Ozone reacts with various chemical compounds in aqueous systems in two different and coexisting modes, one involving direct reactions of molecular ozone and the other a free radical-mediated reaction.  Both these mechanisms may be involved in the destruction of bacteria by ozone.
In this study, chronic periodontitis patients were selected as the above-mentioned periodontal pathogens have been observed in both the disease entities.  Owing to the small sample size, chronic and aggressive periodontitis was grouped as periodontitis patients for statistical and discussion purposes.  In this preliminary trial, the primary focus was to evaluate the efficacy of ozone on specific microorganisms present in subgingival plaque with single irrigation of ozone during the short period of 7 days. This short-term study was followed based on a study in which analysis of the survival of selected periodontal pathogens after scaling and rootplaning (SRP) and intrapocket irrigation was performed after 1 week and 1 month. 
There are several ways of delivering chemical agents. One of the ways such as the subgingival irrigation interferes with the complex ecosystem required for the initiation and continued destruction of the compromised periodontium in the susceptible host. The effects of irrigation on gingival bleeding and plaque include change in plaque composition, flushing out of inflammation-inducing factors and physical change in tissue integrity. 
In the present study, the percentage reduction of plaque index was higher in the ozone (12%) group as compared to the CHX (4%) group. The single subgingival irrigation of 0.12% CHX showed significant reduction of Plaque Index at the end of 1 week. ,, The single subgingival irrigation of 0.12% CHX and twice-daily irrigations with 0.12% CHX, while assessing the clinical and microbiological parameters at the end of weeks 1, 2, 4, 6, 8, 10 and 12, demonstrated a reduction in the Gingival Index from baseline to 1 week. This was also in accordance with the study of Lander et al., who had determined the effect of a single subgingival irrigation of 0.2% CHX at baseline and at weeks 1, 2, 3, 4, 5, 7 and 10, and had shown a reduction in the Gingival Index from baseline to 1 week. 
There was significant reduction of Gingival Index (29%) and Bleeding Index (26%) in the ozone group as compared to the CHX group. Another study reported the significant clinical improvement of the periodontal baseline conditions after the repeated subgingival oxygen irrigations in previously untreated periodontal pockets. 
The antibacterial efficacy of ozone using PCR demonstrated the reduction of Aa (50%) whereas no changes were observed for Pg and Tf on the 7 th day. The chlorhexidine group revealed no changes in the presence of Aa, Pg and Tf. A study that tested the efficacy of intrapocket irrigation with CHX 0.12% and SRP at baseline, 1 week and 1 month, on clinical parameters and microbiological profile, demonstrated a reduction in the percentage of sites that harbored Aa and Pg from baseline to 1 week.  Similar reduction in Aa and Pg was reported using CHX chip. 
Recently, it has been shown that ozonated water was highly effective in killing both Gram-positive and Gram-negative oral microorganisms like Streptococci, Pg, P. endodontalis and Candida albicans in vitro. The number of viable S. mutans remarkably decreased when treated with ozonated water and it also inhibited the accumulation of experimental dental plaque in vitro. When the dental plaque samples from human subjects were exposed to ozonated water in vitro, almost no viable bacterial cells were detected. Hence, it was suggested that ozonated water should be useful in reducing the infections caused by oral microorganisms in dental plaque.  Moreover, the susceptibility of bacterial phenotypes in biofilms is different to those planktonic phenotypes. 
The effect of ozone on Candida albicans (12.5%) was noticeable as compared to chlorhexidine, which did not demonstrate an antifungal effect. The viable Candida albicans were nearly non-existent after the application of ozonated water (2-4 mg/l) for 1 min on the denture plates.  This study result is not in agreement with the study results of Ellepola et al., which showed that CHX had an anti-Candidal effect. 
The antiviral effect of chlorhexidine was pronounced in terms of %reduction of HCMV (12.5%) and EBV (0%) as compared to ozone. In the present study, ozone did not demonstrate the antiviral effect. Bernstein et al. assessed the in vitro virucidal effectiveness of 0.12% CHX mouthrinse on several viruses of the oral cavity and showed that 0.12% CHX had shown a reduction of HSV-1 and HCMV when exposed for 30 s, 5 min and 15 min. 
The percentage of occurrence of Pg, Tf and HSV-1 was higher on the fourth day, which returned to baseline value on the 7 th day, using ozone irrigation. In the chlorhexidine group, the percentage of Pg was higher, which, however reached baseline levels on the 7 th day, but the percentage of Tf and EBV was lower on the 4 th day and reached the baseline levels.
The possible explanation of variation in occurrence of microorganisms during study trial (4 th -7 th day) could be attributed to the antibacterial activity of the agent used. Secondly, the PCR technique can detect both viable and non-viable bacteria. Thus, the diagnostic importance of PCR is immeasurable. If the non-viable microorganisms are detected following antimicrobial therapy then the effectiveness of the antimicrobial agent is difficult to assess, i.e. prognostic value of PCR is compromised because it detects even non-viable organisms. In such a case, the culture method is still the gold standard.
In the present trial, ozonated water irrigation showed a similar effect as compared to the widely used chlorhexidine technique in periodontitis. The plaque, gingival inflammation and bleeding reduction were higher in the ozone group. The antibacterial and antifungal efficacy of ozone on Aa and Candida albicans was appreciable. No antiviral property of ozone was observed. The antiviral efficacy of chlorhexidine was better than that of ozone. Despite the substantivity of chlorhexidine, the single irrigation of ozone is quite effective. Ozone has shown better effects than chlorhexidine, except for the antiviral property. The disadvantages of CHX such as taste alteration and staining are potential drawbacks of chlorhexidine. Although PCR was a choice in this study, the culture technique should be used further so that live bacterial estimation should be fruitful.  Considering the limitation of this study in terms of short-term duration, ozone can be considered as a promising antimicrobial agent in the periodontal therapy. Further long-term studies are required to adequately assess the efficacy of ozone in vivo and to evaluate the frequency and duration of application of ozone. It is required to determine the specific ozone concentration that is effective against anaerobic periodontopathogens.  Because the purpose of this preliminary trial was to study the effect of ozone on few of the periodontopathogens from active deep periodontal pockets of periodontitis patients, pooled plaque samples were collected from the selected sites. , However, site-specific studies are more relevant. It can be concluded that local ozone application can serve as a potential agent to treat periodontal disease non-surgically both for home care and for professional practice. It may serve as a good tool during supportive periodontal therapy.
The one drawback of ozone is its unstable nature. The absorbance of ozone in the water increased almost linearly with time, from 5 to approximately 60 s (data not shown). The stability of ozone in the water was low and ozone dissipated very quickly in ozone demand free water at room temperature over 5 min, which was in agreement with Shechter. ,
| Conclusion|| |
As an alternative management strategy, ozone may be considered due to its powerful ability to inactivate microorganisms. There is growing evidence that it can be employed as a useful therapeutic agent to treat bacteria-, virus- and fungus-associated periodontitis both for home care and at the professional level.
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Vandana K Laxman
Department of Periodontics, College of Dental Sciences, Davangere, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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