Efficacy of Probiotics Compared to Chlorhexidine Mouthwash in Improving Periodontal Status: A Systematic Review and Meta-Analysis

Purpose To critically evaluate the available literature and conduct a systematic review of recent randomized controlled trials to assess the effectiveness of probiotics compared to chlorhexidine mouthwash in enhancing periodontal health. Methods Five databases were searched electronically, as well as the gray literature. Using the Cochrane risk-of-bias tool for randomized clinical trials, the risk of bias was examined. The weighted mean difference (WMD) method was used to calculate the effect sizes. Heterogeneity was assessed using I2 and τ2 statistics. The GRADE approach was adopted to assess the certainty of the evidence. To assess the robustness of the findings, sensitivity analysis and publication bias assessment were undertaken. Results A total of 1850 studies were initially identified. Sixteen clinical trials were eligible for qualitative synthesis, and ten were included in the meta-analysis. In terms of the gingival index, in total, no statistically significant difference was observed between chlorhexidine and probiotics within 4 weeks (WMD −0.03, 95% CI: −0.09∼0.04, P = 0.3885). Similar to GI, no statistically significant difference was observed between chlorhexidine and probiotics regarding the plaque index within 4 weeks (WMD 0.11, 95% CI: −0.05∼0.28, P = 0.1726). No statistically significant difference was observed between chlorhexidine and probiotics in all time intervals regarding oral hygiene index-simplified (WMD −0.01, 95% CI: −0.05∼0.04, P = 0.7508). The robustness of these findings was confirmed by sensitivity analysis and publication bias assessments. Conclusions Based on the findings, probiotics were an acceptable alternative to conventional chlorhexidine in improving periodontal health. High-quality studies with rigorous methodology should be conducted to assess the optimum doses of probiotics for clinical implications.


Introduction
One of the most prevalent oral diseases in the world is periodontal disease [1]. Dental plaque has been proved to be the main factor in the onset and progression of periodontal diseases. Tus, the prevention and treatment of periodontal diseases are based on plaque control and antimicrobial therapies [2]. control is chlorhexidine, a broad-spectrum antimicrobial agent [2]. However, continuous use of chlorhexidine has side efects such as an increase in calculus formation, alteration in taste, and oral mucosal erosion [4].
Probiotic usage has been suggested as an alternative to manage periodontal diseases in recent years. In this way, probiotics may be a turning point in periodontal treatment [5]. Tey were defned as "living microorganisms that, given in sufcient quantities, bring health benefts to the host" [5]. Probiotics have been shown to reduce the acidic pH inside the oral cavity and release bacteriocins that prevent plaque formation [6].
Moreover, previous studies have shown that the use of probiotic products can reduce oral caries. In these studies, reducing the level of some bacteria efective in causing caries, including Streptococcus mutans, has been shown [7]. Probiotics have also been reported to be associated with decreased Candida colonies in saliva and the prevalence of oral candidiasis. By inhibiting the growth of microorganisms, probiotics can change the host's microbiome [8].
Te role of probiotics in periodontal disease and a signifcant decrease in plaque indices, bleeding on probing, and gingivitis have been proposed [9]. However, only a small number of clinical trials have examined the antiplaque and anti-infammatory efects of probiotics and chlorhexidine mouthwash. Tis systematic review and meta-analysis aimed to compare the use of chlorhexidine mouthwash and oral probiotics to evaluate the efcacy of probiotics as a potential alternative agent for the improvement of periodontal status and critically appraise the available literature.

Protocol and Registration.
Tis review was organized based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [10]. Also, it was registered at PROSPERO with the protocol registration code: CRD42021261054.

Eligibility Criteria.
Te main objective of this metaanalysis was to respond to the following question: Do probiotics have the same periodontal health-improving power as CHX mouthwash?
Te PICO components were the following items: population (any patients with no restrictions regarding their age), intervention (probiotics in any form or type), comparator (conventional chlorhexidine mouthwash), outcome (clinical parameters such as the plaque index, the gingival index, the probing pocket depth, the clinical attachment level, the sulcular bleeding index, gingival recession, and the periodontal infamed surface area index), and study design (randomized controlled trials). No language restriction was applied to decrease the risk of language bias. Studies were excluded if they were nonpeerreviewed RCTs and conference papers, editorial, and review papers.

Study Selection.
Two reviewers (KHS and OBLM) rigorously and impartially evaluated the articles. Reading through all of the article titles and abstracts in the aforementioned databases served as the frst step in the selection process. Articles that did not meet the predefned inclusion criteria were excluded after reading the titles and abstracts. Te full texts of the papers that were retrieved after the frst step were gathered, and full-text publications were evaluated by the authors following inclusion criteria. Any discrepancies concerning eligibility and any disputes between the two reviewers were settled by discussion.

Data Extraction.
Te data were extracted by one author, and a second reviewer subsequently double-checked the data. All the data obtained by the articles were tabulated as follows: study design, sample size, age of participants, experimental groups, comparator groups, clinical parameters, and follow-ups. In addition, if studies had insufcient data for the meta-analysis, the authors were contacted to provide them.
2.6. Risk of Bias. Two reviewers (GMS and KHS) independently evaluated the risk of bias in this review using the Cochrane Collaboration's assessment tool (version 2) [11]. Te randomization procedure, variations from intended interventions, missing outcome data, assessment of the outcome, and selection of the reported result are the fve primary domains of this tool.

Data Analysis.
Te meta-analysis was conducted on the gingival index, plaque index, and oral hygiene indexsimplifed (OHI-S). Te "meta" package and the R software, version 3.6.2, were used for all of the analysis. If the data were numerically similar, the weighted mean diference (WMD) utilizing the inverse variance approach was examined for continuous variables. Apart from that, the standard mean diference (SMD) was used. Te fxed-efect model was used when I 2 = 0, and the random-efect model was used when I 2 > 0. All the P values were two-sided, and the statistical signifcance was defned at a level of α = 0.05. Te same methodology used in three previously published meta-analyses was adopted [12][13][14]. Te leave-one-out method, which recalculates the meta-analysisN − 1 time while omitting one study each time, was used for sensitivity analysis. Outliers can be discovered in this method. Re-analysis was performed after the outlier studies' removal to test the robustness of the results. Egger's test [15] and Duval and Tweedie's trim-and-fll method [16] were used to quantitatively assess the publication bias, and contourenhanced funnel plots were built to visualize it.

Certainty Assessment.
Te Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) ranking through fve analysis criteria was used to evaluate the level of the evidence (risk of bias, inconsistency, indirect evidence, imprecision, and publication bias). As a result, the degree of certainty in the evidence was rated as high, moderate, low, or extremely low [17].

Review of the Included Studies.
Te main characteristics of the included studies are presented in Table 1. Te papers compared the use of probiotics (experimental group) in any form with chlorhexidine mouthwash (comparator). Six studies had other experimental groups [18,19,21,26]. Eleven studies had control groups using saline, mint water, distilled water, or regular oral hygiene measures [20,22,23,[25][26][27][28][29][30]33]. Te studies were conducted between 2010 and 2021.829 patients were evaluated. Te number of participants ranged from 15 to 90, divided into experimental and comparator groups, with a mean of 51.8 participants. Te included studies assessed whether daily oral administration of probiotics could infuence the infammatory response and plaque accumulation. Tirteen studies evaluated PI and GI [18-25, 27, 30, 33]. Tree papers evaluated only PI [26,28,30]. Five papers also evaluated OHI-S [18,22,23,30]. Figure 2. Te majority of the included RCTs showed a moderate risk of bias and quality due to deviations from intended interventions [18-25, 27-30, 33]. On the other hand, two showed a low risk of bias [26,33].

Publication Bias Assessment.
Te results of the publication bias assessment are presented in Table 2 and Figure 3. Based on the quantitative tests, publication bias was not proved for all outcomes. However, according to the asymmetric pattern of funnel plots, publication bias was suspected to some extent.

Gingival Index.
In terms of the gingival index, no statistically signifcant diference was observed between CHX and probiotics in all time intervals (WMD −0.03, 95% CI: −0.09∼0.04, P � 0.3885) with a very low level of evidence ( Figure 4 and Supplementary Table 2). After removing outlier studies, similar to the previous analysis, no signifcant diference was observed in all time points (WMD −0.05, 95% CI: −0.11∼0.01, P � 0.0971) with a very low level of evidence (Supplementary Figures 1 and 2 and Supplementary  Table 2).

Plaque
Index. Similar to GI, no statistically signifcant diference was observed between CHX and probiotics regarding PI (WMD 0.11, 95% CI: −0.05∼0.28, P � 0.1726) with a very low level of evidence ( Figure 5 and Supplementary Table 2). On the contrary, a statistically signifcant diference was shown in the fourth week after using mouthwashes (WMD 0.16, 95% CI: −0.05∼0.28), which favored chlorhexidine, with a moderate level of evidence ( Figure 5 and Supplementary Table 2). Moreover, after removing outliers, using sensitivity analysis, no statistically signifcant diference was observed between CHX and probiotics regarding PI (WMD 0.01, 95% CI: −0.03∼0.05, P � 0.5272) with a very low level of evidence (Supplementary  Figures 3 and 4 and Supplementary Table 2).

Discussion
Concerning chlorhexidine's long-term side efects, researchers have been looking for an alternative agent to improve the periodontal status and manage periodontal diseases. Tus, probiotics have been the subject of many clinical trials to prove their efcacy in reducing plaque accumulation and gingival infammation [8].

International Journal of Dentistry
After pooling efect sizes, no statistically signifcant diference was observed between probiotics and chlorhexidine mouthwash within 4 weeks of follow-up. Tere was no signifcant statistical diference found in the gingival index. Tis fnding demonstrates that probiotics have been shown to improve infammatory response. It is well known that probiotics contain benefcial commensals, which operate as a natural barrier against bacteria [5][6][7]34]. Probiotics lessen bacterial adhesion to tooth surfaces, which may prevent microbial growth and proliferation as well as the development of the intercellular plaque matrix. It demonstrates the value of utilizing probiotics by altering the biochemistry of plaque, preventing the production of cytotoxic products that alter the ecology of plaque, and preventing toxicities and antibiotic resistance [5][6][7]34].
However, these fndings were based on very low to moderate certainty of the evidence. Te analyses showed very low levels of certain evidence for the gingival index and plaque index outcomes due to a large amount of heterogeneity and the lack of precision. Rücker et al. have proposed three sources of heterogeneity in meta-analyses including clinical heterogeneity (such as diferences between sample characteristics), statistical heterogeneity, and other sources of heterogeneity (such as design-related heterogeneity) [35]. Studies that used various probiotic formulations and   PI 0-14-36 Probiotic mouth rinse and chlorhexidine mouth rinse caused signifcant inhibition of dental plaque accumulation, but probiotic mouth rinse was found to be more efective for inhibition of dental plaque accumulation after 14 days of intervention and 3 weeks after discontinuation of intervention [25] DB-RCT Age, weight, and adherence to oral hygiene advice are a few variables that may have an impact on periodontal health. Moreover, previous research has shown that obesity serves as a potentiator of the periodontal condition because of the elevation of infammatory cytokines [36]. Tese characteristics might have a variety of efects on a heterogeneous sample, making it more challenging for the systematic review's external validity.
Also, the presence of publication bias was suspected based on the asymmetrical pattern of the funnel plots for outcomes. However, publication bias was not proved using statistical tests. It is crucial, however, not to jump to conclusions and interpret the funnel plot cautiously. In fact, publication bias is just one of many possible reasons for funnel plot asymmetry. Small studies with extremely large efect sizes, between-study heterogeneity, and diferent study designs can lead to funnel plot asymmetry [37]. It is a common fnding that low-quality studies tend to show larger efect sizes because there is a higher risk of bias. Large studies require more investment, so, likely, their methodology will also be more rigorous [37].
As a result, these fndings should be interpreted cautiously. To standardize comparisons, we advise that future randomized clinical studies be conducted using the same probiotic composition and chlorhexidine concentration.
We advise conducting more longitudinal studies and microbiological testing before prescribing probiotics as an antiseptic and antibacterial agent. Due to the inclusion of low to moderate-quality primary papers, this review was constrained. Furthermore, the probiotic and chlorhexidine comparison in these clinical trials lacked standardization. Tey employed several types of probiotics, chlorhexidine formulations, and concentrations. Te aforementioned issues should be considered in future studies.

Conclusion
Probiotics are an alternate option for enhancing periodontal health. It might also serve as a substitute for chlorhexidine mouthwash to avoid any potential negative efects. To determine the ideal doses for clinical implications, additional high-quality research with strict methods should be conducted.

Data Availability
Data are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.