Introduction

Recurrent aphthous stomatitis (RAS) is among the most common diseases of the oral mucosa. It is characterized by recurrent, round, or ovoid ulcers that are painful, surrounded by inflammatory erythematous haloes, and covered with a yellow-grayish pseudomembrane [1]. Its etiology is not precisely known, but several local, systemic, immunologic, genetic, allergic, nutritional, and microbial factors have been proposed to be causative [2]. As Helicobacter pylori is an important risk factor for peptic ulcers, which have similar histologic characteristics as oral ulcers, and both types of ulcers can be treated with wide-spectrum antibiotics such as tetracycline, H. pylori is considered to be a potential factor in the development of RAS [3, 4]. It was reported that persistent inflammation and the complex microbiota in periodontal pockets may provide a suitable environment for the colonization of H. pylori [5]. Additionally, bacterial colonization and persistent inflammation in periodontal pockets are a significant risk factor for periodontal disease. Moreover, some bacterial species found in periodontal pockets were reported to be involved in the development of systemic diseases as a result of systemic inflammation, with an increase in circulating cytokines and mediators, direct infection, and cross-reactivity/molecular mimicry between bacterial antigens and self-antigens [6]. The present study aimed to determine the role of oral H. pylori colonization and periodontal health in the development of RAS.

Materials and methods

Patients

After the study protocol was approved by the Hacettepe University School of Medicine ethics committee and the participants provided written informed consent, data were collected from 38 RAS patients and 43 controls without lesions (n = 43). The study was conducted at the outpatient clinics of the Hacettepe University, School of Medicine, Dermatology Department and Periodontology Department, Ankara, Turkey, between June 2013 and August 2013. The diagnosis of RAS was made according to the history of ≥3 attacks of aphthous ulcer annually and clinical examination. Patients who had active ulcers at the time of examination or established ulcers in previous examinations were also included in the study. The age and gender matched controls consisted of individuals who applied for other than RAS to the dermatology outpatient clinic. Patients and controls that were aged <18 years, pregnant, had diabetes mellitus, had Behçet’s disease, or had any other dermatological diseases with oral mucosal involvement were excluded from the study. Participants with a history of antibiotic or systemic anti-inflammatory drug use during the 4 weeks prior to the start of the study were also excluded. Complete patient anamnesis and the characteristics of RAS were evaluated.

Clinical periodontal parameters

The same periodontist (DK) performed periodontal examination of all the patients and controls. Immediately before the examination, the patients and controls were asked about their oral hygiene practices (usual reason for dental exams, time of last dental exam, and frequency of tooth brushing). Periodontal parameters, including periodontal probing depth (PPD), the plaque index (PI), the gingival index (GI), and the clinical attachment level (CAL), were used to evaluate the participants’ periodontal health status [7, 8]. PPD which is the distance from the gingival margin to the base of the gingival sulcus and CAL which is the distance from the cementoenamel junction to the base of the gingival sulcus were measured (mm) using a William’s periodontal probe. PPD and CAL scores were recorded for six tooth surfaces (mesiobuccal, mid-buccal, distobuccal, mesiolingual, mid-lingual, and distolingual) for all teeth. The numerical scores were calculated according to the following formulae:

$$ \mathrm{Score}\;\mathrm{per}\;\mathrm{tooth}\left(\mathrm{P}\mathrm{P}\mathrm{D},\;\mathrm{C}\mathrm{A}\mathrm{L}\right)=\frac{\mathrm{sum}\;\mathrm{of}\;\mathrm{individual}\;\mathrm{tooth}\;\mathrm{surface}\;\mathrm{scores}}{6} $$
$$ \mathrm{Score}\;\mathrm{per}\;\mathrm{per}\mathrm{son}\left(\mathrm{P}\mathrm{P}\mathrm{D},\;\mathrm{C}\mathrm{A}\mathrm{L}\right)=\frac{\mathrm{sum}\;\mathrm{o}\mathrm{f}\;\mathrm{the}\;\mathrm{scores}\;\mathrm{f}\mathrm{o}\mathrm{r}\;\mathrm{each}\;\mathrm{tooth}\;}{\mathrm{number}\;\mathrm{o}\mathrm{f}\;\mathrm{teeth}} $$

PI and GI scores were evaluated according to a 4-point scale for four tooth surfaces (mesial, distal, buccal, and lingual) [7, 8]. The scores were calculated for individual teeth and participants separately, according to the following formulae:

$$ \mathrm{Score}\;\mathrm{per}\;\mathrm{tooth}\left(\mathrm{PI},\;\mathrm{G}\mathrm{I}\right)=\frac{\mathrm{sum}\;\mathrm{of}\;\mathrm{individual}\;\mathrm{tooth}\;\mathrm{surface}\;\mathrm{scores}}{4} $$
$$ \mathrm{Score}\;\mathrm{per}\;\mathrm{per}\mathrm{son}\left(\mathrm{PI},\;\mathrm{G}\mathrm{I}\right)=\frac{\mathrm{sum}\;\mathrm{o}\mathrm{f}\;\mathrm{the}\;\mathrm{scores}\;\mathrm{f}\mathrm{o}\mathrm{r}\;\mathrm{each}\;\mathrm{tooth}\;}{\mathrm{number}\;\mathrm{o}\mathrm{f}\;\mathrm{teeth}} $$

Dental plaque collection and detection of H. pylori

A commercially available RUT kit (Strong Biotech Corporation, Taiwan) was used to determine the presence of H. pylori. Dental plaque samples were removed from two teeth with the deepest periodontal pockets using a sterile periodontal curette. Both supragingival and subgingival plaque were collected from the tooth surfaces, then immediately inoculated onto the RUT kit paper. The test kits were stored at room temperature, and test paper color change was checked 24 h later; yellow was considered negative, whereas pink or magenta was considered as a positive test result.

Statistical analysis

Statistical analysis was performed using SPSS v.21.0 software for Windows (IBM SPSS, IBM Corp., Armonk, New York, USA). Continuous variables are presented as mean ± SD. Categorical variables are presented as frequency and percentage. Continuous variables were checked for parametric test assumptions. The patient and control groups were compared using the independent samples t test or Mann–Whitney U test, as appropriate. The chi-square test or Fisher’s exact test was used to compare categorical variables. Logistic regression analysis was used to determine factors including clinical periodontal parameters and RUT findings affecting RAS. The level of statistical significance was set at P < 0.05.

Results

Demographic findings

This prospective study included 38 RAS patients (19 male and 19 female) with a mean age of 35.11 ± 10.28 years and 43 controls (21 male and 22 female) with a mean age 34.93 ± 11.03 years. There were not any significant differences in age or gender between the groups. The patients’ characteristics are shown in detail in Table 1.

Table 1 Demographics and baseline characteristics of the patients
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Disease characteristics

Characteristics of RAS, including mean duration of disease, mean ulcer healing time, types of ulcers, ulcer localization, frequency of attacks, number of lesions per attack, and precipitating factors for ulcer development, are summarized in Table 2.

Table 2 RAS characteristics
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Clinical periodontal parameters

Among the study participants, 13 (34.2 %) RAS patients and 11 (25.6 %) controls had dental exams for control, 21 (55.3 %) patients and 28 (65.1 %) controls had a dental exam within the previous year, and 33 (86.8 %) patients and 32 (74.4 %) controls brushed their teeth daily. Differences in the reason for dental exams, time of last dental exam, and daily tooth brushing between the RAS patients and controls were not significant, as shown in Table 3. PPD, PI, GI, and CAL scores were lower in the RAS patients than in the controls, but the differences were not significant (P > 0.05 for all).

Table 3 Clinical periodontal parameters and RUT findings in the patient and control groups
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RUT findings

RUT results were positive in 34 (89.5 %) patients and in 24 (55.8 %) controls; the RUT positivity rate in the patient group was significantly higher than in the control group (P = 0.002).

The relationship between RUT, and disease characteristics and clinical periodontal parameters

The relationship between RUT positivity, and disease characteristics and clinical periodontal parameters are presented in Table 4. The differences in the frequency of attacks and number of lesions per attack between the RUT-positive and RUT-negative patients were not significant (P > 0.05, for both). In addition, there were not any significant differences in the reason for dental exams, time of last dental exam, or frequency of tooth brushing between the RUT-positive and RUT-negative patients. PPD, PI, GI, and CAL scores were higher in RUT-positive patients than in the RUT-negative patients, although the differences were not significant (P = 0.05, for all).

Table 4 The relationship between RUT status, and oral hygiene practices and periodontal parameters in the RAS patients
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The results of the logistic regression analysis of the factors associated with RAS are summarized in Table 5. RUT positivity was observed to be a significant risk factor for the development of RAS (P < 0.001).

Table 5 Logistic regression analysis of the factors associated with RAS
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Discussion

Periodontal diseases are inflammatory diseases of the gums and tooth-supporting tissues that are caused by microbial shifts in the oral cavity [9–12]. As such diseases are chronic, inflammatory, and infectious in nature, they have been proposed to play an etiological or modulating role in several chronic systemic conditions, including cardiovascular and cerebrovascular disease, diabetes, respiratory disease, chronic kidney disease, adverse pregnancy outcome, and H. pylori infection [13–18]. It was recently reported that there is a positive correlation between periodontal disease and oral ulcers in patients with Behcet’s disease; periodontal parameter scores in patients with Behcet’s disease were higher than in controls [15–18]. The similarity of the characteristics of RAS and Behcet’s disease ulcers led us to consider that there might be a similar correlation between periodontal disease and RAS.

The present findings show that PPD, PI, GI, and CAL scores were lower in patients with RAS than in the controls, but that the differences were not significant (P > 0.05 for all), which is inconsistent with the findings reported in studies on Behçet’s disease [15–18]. Whereas, RAS is limited to the oral mucosa and tissue-specific autoimmunity is a probable mechanism in its pathogenesis, Behcet’s disease is a systemic inflammatory disease with different pathogenetic mechanisms and may be not involved in RAS [19].

In the present study, there was not a significant difference in the frequency of tooth brushing between the RAS and control groups. The frequency of tooth brushing was high enough in the patient group, despite the pain associated with the ulcers and the effect of the trauma of tooth brushing on the development of RAS. Logically, it would be expected that RAS patients would avoid tooth brushing due to the pain and trauma, but the present findings indicate otherwise. RAS is a painful disease that can negatively affect quality of life [20]; therefore, we think that the present study’s RAS patients might have been vigilant about their oral hygiene and might have visited their dentists regularly in an effort to mediate such negative effects.

Recent findings concerning the role of H. pylori in RAS are inconsistent [21]. Based on the similarity of the histologic features of gastric and oral ulcers, and the fact that both types of ulcers can be treated with wide-spectrum antibiotics such as tetracycline, H. pylori is suspected to play a role in the in pathogenesis of RAS [3, 4]. Birek et al. [22] suggested that adherence of H. pylori to the oral mucosa and subsequent production of autoantibodies to epitopes shared by oral epithelium cells and H. pylori might result in the tissue destruction associated with RAS. They postulated that H. pylori might be a cofactor in the pathogenesis of RAS, especially in individuals sensitized via gastric colonization and mucosal attachment [22]. In addition, some researchers report that oral H. pylori colonization might arise from gastric colonization via gastroesophageal reflux [23]. In the present study, the observed high prevalence of H. pylori colonization in the RAS patients further indicates that H. pylori plays a role in the pathogenesis of RAS; however, while investigating the role of H. pylori colonization, it was not determined if the source of H. pylori was a permanent reservoir in the oral mucosa or gastric colonization. In addition, such symptoms as dyspepsia and loss of appetite, which can be associated with oral or gastric H. pylori colonization were not investigated; both omissions are limitations of the present study.

There are several methods for detecting H. pylori in gastric mucosa [24]. Studies have reported that RUT has a specificity near 100 % and sensitivity between 70 and 90 % in gastric biopsy samples [25, 26]. In the present study, we found positivity of RUT in 89.5 % of the patients and in 55.8 % of controls. Although urease tests are reasonably specific for detection of the microorganism in gastric biopsy specimens, investigators have doubted its reliability for detecting H.pylori in oral specimens because of other urease-producing bacteria, including Streptococcus vestibularis and Actinomyces viscosus [27, 28]. As such, we think the present findings must be confirmed based on more sensitive techniques such as polymerase chain reaction (PCR). In the present study, H. pylori was detected in dental plaque samples obtained during dental examinations. H. pylori has also been isolated from saliva and the oral mucosa via swabbing [5]; as such, we think it may be practical to routinely collect samples via swabbing and perform RUT in dermatology outpatient clinics.

In the present study, there was not a relationship between RUT positivity and the severity of RAS, according to the frequency of attacks and number of lesions per attack. In addition, H. pylori colonization was associated with RAS, but not the severity of RAS, but we think more research is warranted in order to clarify the relationship between oral H. pylori colonization and the severity of RAS.

In addition to investigating the role of H. pylori colonization in the development of RAS, the present study evaluated its effect on periodontal parameters in RAS patients. All clinical periodontal parameters were higher in the present study’s RUT-positive patients than in the RUT-negative patients, but the differences were not significant. According to the literature, the precise role of H. pylori colonization in periodontal disease remains unknown, but numerous studies have reported H. pylori colonization in 5.9–79 % of subgingival plaque samples in patients with periodontitis [29–32]. In contrast to reports of the role of H. pylori colonization in periodontal disease, Namiot et al. [33] reported that there is not a correlation between dental plaque H. pylori antigen and the number of natural teeth, carious teeth, filled teeth, the plaque index, or the periodontal index. Okuda et al.[34] reported that Streptococcus mutans and Prevotella intermedia inhibited H. pylori growth in the oral cavity, which led us to hypothesize that the increase in the populations of these bacteria in cases of periodontal disease might inhibit oral H. pylori colonization, but the present findings did not support the hypothesis.

In conclusion, the present findings indicate that there might be an association between H. pylori and RAS, but periodontal parameters have no effect on the development of RAS. The small sample size of 38 patients and short inclusion period of 3 months are limitations of the study. Additional studies, with larger sample sizes and longer inclusion periods, are required to more clearly understand the correlations between H. pylori and RAS and periodontal disease.