The efficacy of probiotics in management of recurrent aphthous stomatitis: a systematic review and meta-analysis

There is currently a lack of effective drugs to cure recurrent aphthous stomatitis. This study aimed to evaluate the efficacy of probiotics alone or as an adjunct in recurrent aphthous stomatitis (RAS) patients. Seven randomized controlled trials (RCTs) were included, of which three were included in quantitative analysis. Of five studies evaluating the efficacy of probiotics alone compared with placebo or Oracure gel, two reported no significant difference in relieving oral pain, while probiotics exhibited a higher capacity for decreasing oral pain in the other three. A significant decrease in ulcer severity was found in one, while no significant difference was found in the other four. The remaining two studies demonstrated that probiotics, as an adjunct to steroids or anaesthetic antiseptic gel, significantly reduced the ulcer severity and oral pain. The meta-analysis showed a significant decrease in oral pain (− 1.72, P = 0.0001) with probiotics compared with placebo. In conclusion, probiotics alone were capable of relieving oral pain but not effective in reducing ulcer severity. A combination of probiotics and steroids or anaesthetic antiseptic gel was more effective than steroids or anaesthetic antiseptic gel alone in RAS patients. Probiotics are promising for the treatment of recurrent aphthous stomatitis.

if administered in sufficient amounts 15 . Probiotics play an essential role in systemic health, affecting the immune system and the host's intestinal epithelium to regulate the function of corresponding organs 16 . Furthermore, probiotics (mainly Lactobacilli and Bifidobacteria) have also been introduced to oral medicine in recent years. Many studies have already assessed the efficacy of probiotics in preventing caries, gingivitis, cancer therapy-induced oral mucositis, periodontitis, and peri-implant diseases, which are related to bacterial biofilm and host immune response. The results support the potential use of probiotics in managing gingivitis, cancer therapy-induced oral mucositis, and periodontitis [17][18][19][20] .
As the RAS is associated with bacterial factors and host immune reactions, probiotics have been suggested to have the potential to treat RAS. In 2012, RAS was successfully managed by Lactobacillus brevis CD2 in an immunocompromised hemophilic patient after the failed attempt to use antiviral agents and topical steroids; the condition did not recur for 6 months 19 . And probiotics were proposed as a treatment for oral aphthous stomatitis in patients with inflammatory bowel disease 21 .
In recent years, some clinical studies have explored the efficacy of probiotics in decreasing ulcer severity and alleviating oral pain in RAS patients. However, due to the conflicting results, the efficacy of probiotics is still controversial. Thus, this systematic review and meta-analysis aimed to investigate the effects of probiotics alone or as an adjunctive treatment on RAS patients.

Results
Literature search. Figure 1 presents the search strategy outline in a flow diagram. In the initial search, 147 articles were retrieved from the databases, 87 of which remained after eliminating duplicates. After two independent authors (L.S.Y. and C.B.) read the titles and abstracts, eleven studies remained, which were read in full text. six of the eleven studies fulfilled the criteria in the review and were subjected to data extraction. One additional study was found by conducting a manual search of the reference lists. Finally, seven studies were included in the systematic review for qualitative analysis, of which three studies were included in the quantitative analysis. Table 1 summarizes the characteristics of all the included studies.

Description of the included studies.
Seven studies were published between 2011 and 2020, of which two studies published in 2020 and two in 2017, and the other three were published in 2011, 2014, and 2019 respectively. All the seven included studies were randomized controlled trials. The sample size ranged from 19 to 80 participants. Four studies 22-25 described the age range of the patients, ranging from 8 to 60 years, two studies 26,27 recorded the mean age that was 55.9, 28.2 in the test group and 36.4, 29.38 in the control group respectively, while the one 28 did not mention. Aswath et al. (2014) 28 did not report the gender distribution of the subjects. Three studies 23,25,26 reported the drop-out numbers as 1, 4, and 15, respectively, but there were none in the four other studies 22,24,27,28 . Nirmala et al. (2017) 24 assigned patients to the AU and BU groups for recurrent aphthous ulcers. Patients were treated with oral probiotics as an adjunct to triamcinolone in the AU group and with a triamcinolone paste in the AC group. Aswath et al. (2014) 28 advised patients in test group take anaesthetic antiseptic gel along with bifilac lozenge composed of probiotics while patients in control group take anaesthetic antiseptic gel only. In the five other studies, the intervention in the experimental group consisted of probiotics only, while the control group subjects received a placebo 22,[24][25][26][27] . Patients in studies by Mimura et al. (2017) 23 25 gave patients a combination of two. Some other studies adopted a single probiotic treatment 22,24,26,27 . Concerning the outcome measures, the evaluation assessment for the severity of ulcers are diverse and will be described in detail later. Three studies 23,25,26 using the visual analog (pain) scale (VAS) for the evaluation of oral pain were included in the meta-analysis; four studies 22,24,27,28 were qualitatively described.
Quality of the included studies. The Cochrane Collaboration's tools for assessing the risk of bias and study quality were used to assess the quality of research. Figure 2 presents the detailed results of the assessment. All the seven studies were unclear. The method for generating random sequences in three studies was unclear (Nirmala et  Study outcomes. Effect on ulcer severity. Table 2 presents detailed results on ulcer severity. All the included studies evaluated the severity of ulcers before and after treatment, but the specific content and methods of the assessment were not the same. Dugourd et al. (2020) 26 determined the average decrease in canker sore counts, and the results were not significantly different between the two groups after 90 and 180 days.  www.nature.com/scientificreports/ regarding the ulcer counts because of significantly different baseline characteristics, different interventions, and high heterogeneity.
Effect on oral pain. Table 3 presents detailed results on oral pain. Six of the included studies used the effect on oral pain as an outcome indicator, but different assessment methods and follow-up periods were adopted. Three studies 23,25,26 using VAS to evaluate oral pain related to the RAU were included in the meta-analysis, but all were described qualitatively below.  24,27 reported that probiotics, as a local adjunct, could significantly decrease oral pain compared with control group in the follow-up. With regard to the efficacy, four studies [22][23][24]27 reported that probiotics exhibited a stronger capacity for reducing pain, while the other two 25,26 found no significant differences between probiotics group and placebo group. A meta-analysis regarding oral pain was conducted. The inter-study heterogeneity regarding the oral pain related to RAU was low (χ 2 = 0.83, P = 0.0001, I 2 = 0%) and a fixed-effect model was selected. A higher decrease in oral pain was obtained in favor of probiotics treatment (MD = − 1.72, 95% CI [− 2.59; − 0.85], P = 0.07) (Fig. 3).
Quality of oral health. In addition to oral pain and the severity of the ulcer, Dugourd et al. (2020) 26 assessed patients' QoL in two groups before treatment and after 90 and 180 days with OHIP-14. The results showed no significant differences (P = 0.827). 1 refused to continue and treatment never started, 1 screen failure and treatment never started, 1 lost to follow-up, 1 refused to continue. b Data from day 90 is missing in 1 patient in the test group. c The author didn't mention the reason that why those participants dropped out.  www.nature.com/scientificreports/ 2020) 22,25,26 . The remaining study did not report any side effects.

Discussion
The present study assessed the efficacy of probiotics alone or as an adjunct to other drugs for recurrent aphthous stomatitis (RAS) patients compared with placebo or other drugs alone in terms of the severity of ulcers and oral pain. Only RCTs were included. The overall risk of bias in all the included studies was unclear based on the Cochrane risk-of-bias tool. Due to the high heterogeneity regarding the ulcer severity, a meta-analysis was not performed. The severity of ulcers was qualitatively analyzed, and a meta-analysis was carried out concerning oral pain. Generally, applying probiotics alone was effective in relieving oral pain but not effective in reducing ulcer severity. The results of the meta-analysis showed that higher oral pain relief was obtained in favor of probiotics treatment. Probiotics, as a local adjunct to steroids or anaesthetic antiseptic gel, could significantly reduce the ulcer severity and relieve oral pain. Overall, probiotics have a particular effect on RAS. However, there are still some points worth debate concerning (1) the dose and the application of mono versus mixed species probiotics   25 , Lactobacillus brevis CD2 and Bacillus clausii exhibited an excellent capacity for promoting ulcer healing and reducing oral pain. Different therapeutic effects might be related to different ingredients of probiotics. Future studies should further explore whether Lactobacillus brevis CD2 and Bacillus clausii can outperform other probiotics in treating RAS. The effects of different doses on therapeutic efficacy should also be explored.
As a useful tool for evaluating oral pain, VAS has exhibited high reliability and validity and is more objective than other evaluation methods included in the study. Various methods are available for evaluating the severity of  www.nature.com/scientificreports/ ulcers. At this point, it was difficult to interpret and compare outcomes. Thus, a more standardized scoring system is required to assess the ulcer severity in RAS patients. Future studies also should aim to report on outcomes using a universally accepted evaluation index. The present study showed that the effect of probiotic treatment is closely related to the follow-up time.  27 last for 14 days and 5 days. The effects of probiotic therapy were more effective than the control group. However, for the other three studies, the follow-up periods were more than 90 days 23,25,26 . The results showed that they were not more effective in reducing ulcer severity and oral pain than the control group. In other fields, the duration of probiotic therapy and follow-up has been reported to be a confounder 31 . It can be speculated that probiotics are effective in treating aphthous ulcers in the short term, but the long-term effects are still to be elucidated, necessitating more high-quality studies. No significant side effects were reported in the included studies. Besides, probiotics have exhibited biological safety in treating other diseases, and the safety of probiotic preparations is generally satisfactory. To date, the adverse effects of these probiotic drugs have been considered few, and reports on their harmful effects in the host are rare, except for some people with underlying medical conditions 32 . Currently, Lactobacillus and Bifidobacterium are effectively used to treat some intestinal diseases, such as diarrhea, which are almost the same as drugs used to treat some oral diseases 33,34 . Most probiotic products are marketed as food and given a designation called GRAS (generally regarded as safe) 35 . However, there may still be a systemic infection caused by bacterial translocation, excessive immune response, gastrointestinal adverse reactions, and other side effects 36,37 . Probiotic drugs used in patients with immune deficiency, severe diseases, and gastrointestinal epithelial injury are prone to systemic bacteremia and infection 38 . Probiotics might increase mortality in patients with severe acute pancreatitis 39 . Lactobacillus preparation might cause lactic acidosis and increase the risk of sepsis, meningitis, and other diseases 40 . Therefore, the application of probiotic preparations should be carefully observed to prevent disease progression. More studies are needed to elucidate the potential adverse effects of probiotic treatment.
All the included studies were RCTs, which ensured the credibility of the results. Also, we focused on applying probiotics in the treatment of oral ulcers, which might provide new ideas for treating oral ulcers in clinical practice. To the best of our knowledge, this article is the first systematic review to focus on the effect of probiotic drugs in RAS. However, this study had some drawbacks.  23 mentioned that they used double-blind in participants and personnels. Thirdly, we still need more samples and studies to confirm the credibility of the results. Fourthly, patients' oral hygiene status and microecological environment were not recorded, and whether the microbial distribution is correlated with the treatment effect is worth further exploration.
Due to the limited evidence of current studies, more high-quality studies with a better methodology are necessary before the final clinical application. A crossover design is considered more suitable for clinical trials on RAS, which is a recurrent, stable and agnogenic disease, than a parallel design. With this study design, on the one hand, every participant is compared to themselves, thus eliminating the influence of nonprocessing factors on the results, including sex, age, oral hygiene habits, oral health, general health, and smoking. If a parallel design is adopted, there should be no significant difference in these nonprocessing factors between the experimental and control groups. On the other hand, a smaller sample size is required to reach sufficient statistical power compared with parallel trials. A better study design is comprised of larger sample size, more reasonable randomization, more rigorous blinding, fewer missing outcome data, and more reasonable evaluation intervals, including shortterm and long-term intervals. Currently, some clinical studies are underway, with only a few of them comparing the efficacy of probiotics with other therapeutic methods. Thus, future research should compare the effects of probiotics with mainstream drugs, such as steroids. Probiotics exhibited high efficacy in patients with low immunity 41 . Clinical trials in such patients might have important implications. Finally, the relationship between RAS activity and critical bacteria should be investigated concerning the development and improvement of RAS.

Methods
Focused question. This systematic review was carried out following the Cochrane Handbook for the Systematic Reviews of Interventions and the preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 42 .
The focused PICO question was: Are probiotics alone or as an adjunct to other drugs more effective than placebo or other drugs alone in decreasing ulcer severity and oral pain in RAS patients?
The PICO principle was applied during the assessment. Population/patient: Patients with recurrent aphthous ulcers (RAU). Intervention: Treatment with probiotics, either alone or combined with other drugs. Comparison: Treatment with placebo or other drugs alone.
Outcome: The severity of ulcer or oral pain.
Selection criteria. The inclusion criteria consisted of (1) randomized controlled trials (RCT); (2) assessing the treatment of patients with recurrent aphthous ulcers; (3) comparing probiotics alone versus placebo or probiotics combined with other drugs versus other drugs alone. The exclusion criteria consisted of (1) in vitro or animal studies, case reports, and case series, review articles, or opinion articles; (2) participants with any systematic disease or if they were duplicated or ancillary studies; (3) studies that did not include outcomes required in this review; and (4) studies that were still in process. After eliminating duplicates, two independent authors (L.S.Y. and C.B.) read the titles and abstracts derived from the electronic search to identify potentially eligible articles. If they conformed to the proposed theme, they were assessed in the full text and further evaluated for inclusion criteria. The references of all the included articles or relevant reviews were also considered to avoid missing any eligible studies. Any discrepancies between the authors were resolved by discussion.

Scientific
Data extraction. Three sheets were designed to extract the characteristics and numerical data of the included studies. Two researchers (L.S.Y. and C.B.) separately extracted the data, which included the following: study author, study design, the number of patients, mean age, the diagnosis of the patients, interventions, comparison, the number of patients who dropped out, evaluation index, evaluation interval, follow-up periods, primary outcomes, and conclusions. The evaluation parameters for the severity of ulcer and oral pain were reported using means standard deviations (SD), and P-values. If data in the original articles were absent, the corresponding author was contacted for missing information. Any disagreement between researchers was resolved by discussion. If no agreement could be reached, a third researcher (Z.X.Y.) joined the discussion.

Assessment of risk of bias and studies' quality. The risk of bias was assessed according to the Cochrane
Collaboration's tools 43 . Two independent researchers conducted evaluations without interference. The main criteria for evaluation were the generation of random sequences, whether the assignment was hidden, whether the researchers and subjects were double-blinded, whether the study outcomes were blindly assessed, whether the results were complete, and whether the results were selectively reported. We also designed an additional risk of bias tables to assess other biases, such as whether the article-publishing organization was associated with medication manufacturers or not. Revman manager 5.3 was used for data visualization. If the two researchers' assessments were not consistent, a third researcher reassessed the disputed results or conducted a team discussion.
Statistical analyses. The level of heterogeneity was assessed by the Cochrane χ 2 and I 2 test. P-value of χ 2 test < 0.1 or I 2 > 50% was considered an indicator of high heterogeneity. I 2 > 0.7 was considered too high to conduct a meta-analysis. The fixed-effect model was selected if P > 0.1 or I 2 < 50%, while the random-effect model was selected if P < 0.1 or I 2 > 50%. Mean difference (MD) and standard deviation (SD) were used to perform the meta-analysis. Weighted mean difference (WMD) and 95% confidence interval (CI) were selected as measure estimates of differences between the probiotic and placebo groups. All the statistical analyses were conducted using Review Manager 5.3.

Conclusion
Probiotics alone were effective in relieving oral pain but not effective in reducing ulcer severity. The combination of probiotics and steroids or anaesthetic antiseptic gel was more effective than steroids or anaesthetic antiseptic gel alone in decreasing ulcer severity and oral pain for RAS patients. Probiotics are promising for the treatment of recurrent aphthous stomatitis. However, due to the limited data and study quality, more well-designed clinical studies are necessary.

Data availability
All the data were included in the paper and supplementary information. www.nature.com/scientificreports/