Could polyhexanide and chlorine dioxide be used as an alternative to chlorhexidine? A systematic review

ABSTRACT BACKGROUND: Maintenance of oral microbiota balance is the simplest way to prevent infectious oral diseases, through controlling dental biofilm. Combined use of mouthwash and mechanical removal has been shown to be a very effective way for this. OBJECTIVES: To identify clinical studies comparing the antimicrobial effect and possible adverse effects and/or side effects of chlorhexidine-based mouthwashes with those of mouthwashes containing chlorine dioxide and/or polyhexanide, for controlling oral microbiota. DESIGN AND SETTING: Systematic review designed by the stomatology sector of postgraduation in applied dental sciences of Bauru Dentistry School, University of São Paulo, Brazil. METHODS: A systematic review was conducted using online databases (PubMed, Embase, Web of Science and Science Direct) up to April 8, 2020. The search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: The studies included comprised eight articles published between 2001 and 2017. A total of 295 young adults, adults and elderly people were evaluated (males 44.75% and females 55.25%). Three articles compared polyhexanide with chlorhexidine and five articles compared chlorine dioxide with chlorhexidine. No studies comparing all three mouthwashes were found. The concentrations of the study solutions were quite varied, and all rinses had an antimicrobial effect. In four studies, it was stated that no side effects or adverse effects had been found. Three studies did not address these results and only one study addressed side effects and/or adverse effects. CONCLUSION: Mouthwashes containing chlorine dioxide and polyhexanide are viable alternatives to chlorhexidine, since they reduce oral biofilm and have little or no reported side or adverse effects.


OBJECTIVES
The aim of this systematic review was to identify clinical studies that compared the antimicrobial effect and possible adverse and/or side effects of CHX-based mouthwashes with those of mouthwashes containing ClO 2 and/or PHMB, for controlling dental biofilm.

Research question
Two research questions were formulated: • Do mouthwashes containing PHMB and/or ClO 2 have antimicrobial efficacy in the oral microbiota comparable to that of CHX?
• Do studies with mouthwashes containing PHMB and/or ClO 2 show adverse and/or side effects, in comparison with to the effects associated with CHX?

Study design
This systematic review was conducted in accordance with the PRISMA guidelines (Preferred Reporting Items of Systematic Reviews and Meta-Analyses). 9

Systematic review protocol
The protocol for this systematic review was registered in PROSPERO (CRD42019115929) and is available on the website www.crd.york.ac.uk/PROSPERO/.

Search strategy
A search of the literature was conducted to survey clinical studies that aimed to investigate the antimicrobial action of mouthwashes containing PHMB and ClO 2 , compared with that of CHX.
The studies included were identified based on a search strategy for each electronic database: PubMed, EMBASE, Web of Science and Science Direct. The search strategy was designed with Boolean operators (AND/OR) to identify all studies on this topic published in English, Portuguese or Spanish up to December 21, 2020. The descriptors used were "Chlorhexidine", "Polyhexanide", "Dioxide Chlorine" and "Mouthwash". The search strategies are detailed in Table 1.
In the Science Direct database, filters for research articles (31) and conference abstracts (2) were activated in order to exclude texts from encyclopedias, book chapters and other sources.

Eligibility criteria
This review included clinical studies that evaluated the effectiveness of mouthwashes and studies that compared the action of PHMB and/or ClO 2 in relation to CHX, regardless of the participants' age, sex, systemic changes or medication use.
The following types of studies were excluded: literature review articles, clinical cases or case series, studies that did not evaluate mouthwashes, studies not related to dentistry, in vitro, in situ and animal studies and studies published in other languages.

Data sources, study selection and data extraction
All records collected were moved to a folder of the reference manager EndNote Web (www.myendnoteweb.com). Any duplication of references was identified and then deleted. All studies included were independently examined by two reviewers (D.S.F.S. and F.S.B.) and their main characteristics were extracted in order to perform data synthesis and study quality assessment. Only the information described in the articles was considered.

Data analysis
A narrative data synthesis was carried out, structured around the characteristics of each study, i.e. the microbiological count, type of microorganism, characteristics of the population, parameters evaluated and results obtained.

Risk of bias
Two reviewers (D.S.F.S. and F.S.B.) independently assessed the risk of bias in the studies included through using the Cochrane risk of bias tool (RoB 2.0, 2008), which is available in the Cochrane manual for developing systematic intervention reviews, version 5.1.0 (Cochrane Handbook, Oxford, United Kingdom, and Melbourne, Australia). 10 Any discrepancies were resolved by a third reviewer (P.S.S.S.). This tool was chosen to assess the risk of bias in randomized clinical trials 10 in terms of seven domains: generation of random sequence, allocation concealment, blinding of participants and professionals, blinding of outcome evaluators, incomplete outcomes, selective outcome report and others. These were classified as presenting "low risk", "high risk" or "uncertain risk", in accordance with each criterion of the tool. 10 Afterwards, the data were inserted into the Review Manager (RevMan Version 5.3, Cochrane Manager Review Center, Oxford, United Kingdom) software, and a riskof-bias graph was generated.

Study selection
A total of 245 studies were initially identified in the following databases: PubMed (n = 132), Embase (n = 41), Web of Science (n = 39) and Science Direct (n = 33). Thirty studies were excluded due to duplication. Among the remainder, 48 studies were selected for reading the title and abstract and 39 of these were excluded for the following reasons: they were in vitro or in vivo studies, did not use CHX as a control (comparison), did not use PHMB and/or ClO 2 as an intervention or did not use mouthwashes. Thus, the full texts of nine studies were read. From this, one further study were excluded because it did not meet the eligibility criteria (it was an in vitro and in vivo study about decolonization rates of Staphylococcus aureus). Hence, the final analysis was conducted on eight studies. The detailed sequence can be seen in the study selection flowchart 9 (Figure 1). Table 1 shows the general characteristics of the studies included, which were published between 2001 and 2017. The search was carried out without restriction on publication date. These studies were conducted in Europe and Asia (Germany, Switzerland, Turkey, India and Indonesia). All of them were randomized clinical studies, and microbiological analyses were performed. 8,[11][12][13][14][15][16][17] In total, 295 individuals were evaluated and, in Embase (('chlorhexidine mouthwash'/exp OR 'chlorhexidine mouthwash' OR (('chlorhexidine'/exp OR chlorhexidine) AND ('mouthwash'/ exp OR mouthwash)) OR 'chlorhexidine'/exp OR chlorhexidine OR 'chlorhexidine gluconate'/exp OR 'chlorhexidine gluconate' OR (('chlorhexidine'/exp OR chlorhexidine) AND ('gluconate'/exp OR gluconate))) AND (phmb OR 'polyhexamethylene biguanide'/exp OR 'polyhexamethylene biguanide' OR (polyhexamethylene AND ('biguanide'/exp OR biguanide)) OR polyhexamethylenbiguanid OR 'polihexanide'/exp OR polihexanide OR 'dioxide chlorine' OR (('dioxide'/exp OR dioxide) AND ('chlorine'/exp OR chlorine)) OR 'chlorine dioxide'/exp OR 'chlorine dioxide' OR (('chlorine'/exp OR chlorine) AND ('dioxide'/exp OR dioxide))) AND ('mouthrinse'/ exp OR mouthrinse OR 'mouthwash'/exp OR mouthwash OR 'mouthwashes'/exp OR mouthwashes OR 'mouth bath' OR (('mouth'/ exp OR mouth) AND ('bath'/exp OR bath)) OR 'mouth rinse'/exp OR 'mouth rinse' OR (('mouth'/exp OR mouth) AND rinse) OR 'mouth wash'/exp OR 'mouth wash' OR (('mouth'/exp OR mouth) AND wash) OR 'bath, mouth' OR (bath, AND ('mouth'/exp OR mouth)) OR 'baths, mouth' OR (baths, AND ('mouth'/exp OR mouth)) OR 'mouth baths' OR (('mouth'/exp OR mouth) AND ('baths'/exp OR baths)) OR 'mouth rinses'/exp OR 'mouth rinses' OR (('mouth'/exp OR mouth) AND rinses) OR 'rinse, mouth' OR (rinse, AND ('mouth'/exp OR mouth)) OR 'rinses, mouth' OR (rinses, AND ('mouth'/exp OR mouth)) OR 'wash, mouth' OR (wash, AND ('mouth'/exp OR mouth))))

Science Direct
(Chlorhexidine OR Chlorhexidine gluconate) AND (phmb OR polyhexamethylene biguanide OR polyhexamethylen biguanid OR polihexanide OR chlorine dioxide) AND (mouthrinse OR mouthwash OR mouthwashes OR Mouth Bath OR Mouth Rinse OR Mouth Wash)

Evaluation profile of the clinical trials
One study evaluated the antifungal effects of ClO 2 compared with those of CHX; 14 one compared ClO 2 with CHX, against the chromogenic bacterium species Actinomyces; 16 three evaluated the effect of PHMB compared with CHX, on oral biofilm; [11][12][13] and three compared the effects of ClO 2 with those of CHX, on oral biofilm. 8,15,17,18 One study evaluated totally edentulous individuals and their dentures, 14 six evaluated the teeth and mucous membranes of young adults, 8,[11][12][13]15,17 one evaluated the tongue coating, 8 one evaluated individuals who had undergone orthodontic treatment 17 and one evaluated molar dental sulcus pigmentation in children. 16
In addition to differences in concentrations, there were differences in quantity, duration of exposure to mouthwash solution and duration of the study ( Table 2). The study that evaluated totally edentulous individuals 14 gave the recommendation that individuals should immerse their dentures in the mouthwash, overnight for 15 days.

Study outcomes
The primary outcome from this systematic review was to report on the antimicrobial efficacy of mouthwashes containing PHMB and/or ClO 2 , compared with those containing CHX. The secondary outcome was to report on the adverse effects of mouthwashes.

Antimicrobial efficacy of mouthwashes
All three studies that compared PHMB with CHX used a concentration of 0.12% for CHX. These studies evaluated the action of mouthwashes on bacteria. Among their conclusions, one was that the substantivity of CHX was always 12 hours. [11][12][13] Regarding the biofilm index, the studies showed that there were significantly lower rates with CHX than with PHMB 0.04% (P = 0.038). 11 There were no statistically significant differences between PHMB 0.12% and CHX (P > 0.05), 12 and PHMB 0.2% was significantly less effective on the biofilm index than CHX (P = 0.016). 13 The bacterial count was investigated at two times: four hours after using the mouthwash and five days after this. Evaluation of the bacterial count of the dental surface showed that CHX was significantly more effective in reducing the bacterial count than PHMB 0.04%, at both times evaluated (four hours, P = 0.003; five days, P = 0.030). 11 There was no statistically significant difference between PHMB 0.12% and CHX (P = 0.085) after four hours, while after five days of use, PHMB 0.12% was significantly less effective than CHX (P = 0.008). 12 In the first four hours, with PHMB 0.20%, there was no significant difference compared with CHX (P = 0.623); after five days of use, PHMB 0.2% significantly inhibited bacterial growth, compared with CHX (P = 0.029). 13 Evaluation of bacterial counts on the mucosal surface showed that CHX was significantly more effective than PHMB 0.04% (P = 0.42) 11 and PHMB 0.12% (P = 0.013) 12 after the first four hours and after five days of using PHMB 0.04% (P = 0.007) 11 and PHMB 0.12% (P = 0.000). 12 There were no significant differences between PHMB 0.2% and CHX four hours after use (P = 0.738) or five days afterwards: both solutions were equally effective (P = 1.000). 13 Other studies compared ClO 2 with CHX 8,14-17 and found that CHX 0.2% inhibits biofilm more powerfully than ClO 2 0.01% (P < 0.001). 15 Four hours after use, CHX 0.2% was found to have been more efficient than ClO 2 , such that there were fewer colony-forming units (CFUs) on the mucosa (P < 0.001) and on the dental surface (P = 0.01). 8 Regarding the biofilm index (P = 0.05), rate of accumulation of tongue biofilm (P = 0.238), presence of bacterial CFUs on the fifth day of mouthwash and application of mouthwashes for 15 days, use of ClO 2 was equal to use of CHX 0.2% (P = 0.160). 8 It was concluded that the reductions in the dental biofilm

Adverse effects/side effects
The authors of seven studies 8,11-14,16,17 did not mention the expected adverse or side effects: among these, the authors of four studies reported that they did not observe any adverse effects and/or side effects during their investigations, [11][12][13][14] while such effects were not reported in the results from three studies. 8,16,17 In one other study, 15 a questionnaire regarding the perception of mouthwashes was applied. The participants in that study preferred the taste of ClO 2 over that of CHX (P < 0.001) and reported that there was less change in taste when using ClO 2 than when using CHX (P < 0.001). The taste of CHX remained in the mouth longer than that of ClO 2 (P < 0.001), while use of CHX was more convenient than use of ClO 2 (P < 0.001) and the perception of plaque reduction through using CHX was greater than through using ClO 2 (P < 0.001). 15

Risk of bias
In the present study, the Cochrane risk of bias (RoB) tool 10 was applied to assess the risk of bias in the eight randomized controlled trials that were included. The risk of bias was explored in seven domains.
Two studies were classified as presenting an uncertain risk of bias in three domains, specifically those relating to selection bias (random sequence generation and allocation concealment) and detection bias (blinding of outcome assessment), 14,16 given that in these studies the randomization and allocation methods were not mentioned and it was not reported whether the results were obtained through blind analysis. Six studies were classified as presenting an uncertain risk of bias in relation to detection bias (blinding       of outcome assessment), 8,[11][12][13]15,17 given that it was not addressed whether blinding had been applied in order to obtain the results.
The other domains of all studies were classified as having low risk of bias. No study was classified as having a high risk of bias in any domain (Figure 2). Therefore, overall, the studies included in this systematic review showed good methodological quality (Figure 3).

DISCUSSION
Finding a mouthwash that is as effective as CHX and which has fewer adverse effects has been a challenge for researchers.
In this systematic review, it was seen that in a study that compared PHMB with CHX, the residual antimicrobial action (sub- reduced the amount of tongue coating and Gram-positive and Gram-negative bacteria in the saliva. 27 In dental black spots caused by Actinomyces sp., ClO 2 proved to be statistically more effective in reducing the bacterial viability of Actinomyces sp.
than CHX, after seven days of use. 16 ClO 2 is believed to be an effective alternative for use among children, given that this solution is not carcinogenic or allergenic and does not cause any change in taste sensation. Moreover, there are studies that have suggested that it is less toxic to humans than CHX. 16,28 Therefore, although CHX is typically considered to be the gold standard, ClO 2 is also effective for biofilm control.
When rinses containing ClO 2 and CHX were applied to patients with orthodontic appliances, no statistical differences regarding reduction of the gingival index or total visible biofilm index were  observed. 17 Therefore, the effectiveness of these two solutions for controlling bacterial biofilms seems to be equal.
In a study that evaluated fungal biofilm, 14 a statistically significant reduction in the number of C. albicans hyphae (ClO 2 , P = 0.03; and CHX, P > 0.01) was observed upon treatment with ClO 2 and CHX. Presence of C. albicans in hyphae in the oral mucosa indicated infection by this fungus. 29 The antifungal effects of these two solutions have already been proven. 30,31 In addition to reduction of hyphae, 60% of the patients treated with ClO 2 and 70% of the patients treated with CHX were found to have achieved a cure for inflammation, 14 which thus indicates the antifungal effects of ClO 2 compared with those of CHX.

Limitations
There were some limitations to this systematic review, given that in one study the concentrations of mouthwashes used in the experiment (CHX and ClO 2 ) were not reported 17 and in another the commercial name for the product (Fresh Chlor) was reported but the ClO 2 concentration was not reported. 8 In addition, no study addressed the expected adverse effects. Nor was it reported whether the results from each study were collected in a blinded manner. In this review, no meta-analysis could be performed, given the heterogeneity of purposes observed among the studies included. These conditions also make it difficult to generalize the conclusions, since the synthesis of the results was often based on a limited amount of evidence.

Recommendations
Because the results from the mouthwashes assessed in this systematic review were equal to or more significant than those from the gold standard CHX, 8,[11][12][13][14]16,17 we recommend that future clinical and in vitro studies should be conducted; adverse effects should be considered at the time of evaluation in clinical studies; Eunike et al. 16 Paraskevas et al. 15 Rosin et al. 11 Rosin et al. 12 Uludamar et al. 14 Welk et al. 13 Yadav et al. 8 Yeturu et al. 17 Low risk of bias Unclear risk of bias High risk of bias 0% 25% 50% 75% 100% Random sequence generation (selection bias) Allocation concealment (selection bias) Blinding of participants and personnel (performance bias) Blinding of outcomes assessment (detection bias) Incomplete outcome data (attrition bias) Selective reporting (reporting bias) Other bias

Random sequence generation (selection bias)
Allocation concealment (selection bias) Blinding of participants and personnel (performance bias) Blinding of outcomes assessment (detection bias) Incomplete outcome data (attrition bias)

Selective reporting (reporting bias)
Other bias products should be specified; and blinding of results should be implemented and demonstrated.

CONCLUSIONS
Mouthwashes containing PHMB and ClO 2 are viable alternatives to CHX, since studies showed that the antimicrobial effects of PHMB were comparable with those of CHX and that the antimicrobial effects of ClO 2 were even greater than those of CHX. These alternative solutions have little or no reported side effects or adverse effects. No study compared both PHMB and ClO 2 with CHX.