Recurrent aphthous stomatitis and Helicobacter pylori

Background Recurrent aphthous stomatitis (RAS) is a recurrent painful ulcerative disorder that commonly affects the oral mucosa. Local and systemic factors such as trauma, food sensitivity, nutritional deficiencies, systemic conditions, immunological disorders and genetic polymorphisms are associated with the development of the disease. Helicobacter pylori (H. pylori) is a gram-negative, microaerophile bacteria, that colonizes the gastric mucosa and it was previously suggested to be involved in RAS development. In the present paper we reviewed all previous studies that investigated the association between RAS and H. pylori. Material and Methods A search in Pubmed (MEDLINE) databases was made of articles published up until July 2015 using the following keywords: Helicobacter Pylori or H. pylori and RAS or Recurrent aphthous stomatitis. Results Fifteen experimental studies that addressed the relationship between infection with H. pylori and the presence of RAS and three reviews, including a systematic review and a meta-analysis were included in this review. The studies reviewed used different methods to assess this relationship, including PCR, nested PCR, culture, ELISA and urea breath test. A large variation in the number of patients included in each study, as well as inclusion criteria and laboratorial methods was observed. H. pylori can be detected in the oral mucosa or ulcerated lesion of some patients with RAS. The quality of the all studies included in this review was assessed using levels of evidence based on the University of Oxford’s Center for Evidence Based Medicine Criteria. Conclusions Although the eradication of the infection may affect the clinical course of the oral lesions by undetermined mechanisms, RAS ulcers are not associated with the presence of the bacteria in the oral cavity and there is no evidence that H. pylori infection drives RAS development. Key words:Campylobacter, elisa, h. pylori, Helicobacter Pylori, RAS, recurrent aphthous stomatitis, PCR.


Introduction
Recurrent aphthous stomatitis (RAS) is a very common condition characterized by solitary or multiple small, round, recurrent oral ulcers, with erythematous haloes and circumscribed margins. The appearance of the painful ulcers is periodic and the onset is usually during childhood and tends to diminish in severity with age (1). The diagnosis of RAS is based on clinical grounds but the etiology and pathogenesis remain unclear (2). Local and systemic factors have been suggested to affect the development of RAS. These factors are illustrated in the figure 1. For example, some genetic polymorphisms are associated with the occurrence of RAS (3). Some predisposing factors include trauma, hormonal changes, diet, nutritional deficiencies, Coeliac disease, and immunological disorders (4,5). Regarding nutritional deficiencies, some studies have found decreased levels of iron, vitamin B3 and B12, vitamin C, and folic acid (2). Helicobacter pylori (H. pylori) is a gram-negative, microaerophile bacteria, that colonizes the gastric mucosa and its infection is associated with the development of peptic ulcers, gastric mucosa associated lymphoid tissue lymphoma, and gastric cancer (6). Although H. pylori infection has been suggested to be one of the etiological factors in the pathogenesis of RAS, this association is debatable. In the present paper we review this issue and present the available evidence regarding this controversial topic.

Material and Methods
-Association between RAS and helicobacter pylori In this review, a search in Pubmed (MEDLINE) databases was made of articles published up until July 2015 using the following keywords: Helicobacter Pylori or H. pylori and RAS or Recurrent aphthous stomatitis. We included experimental and review studies that assessed the relationship between H. pylori and RAS. Quality of the studies was assessed using levels of evidence based on the University of Oxford's Center for Evidence Based Medicine Criteria (CEMB 2009) ( Table 1).

Results and Discussion
We included in this review fifteen experimental studies that addressed the relationship between infection with H. pylori and the presence of RAS and three reviews, including a systematic review and a meta-analysis that assessed this association ( Table 2). As shown in table 2 there was a large variation in the number of patients evaluated in each study as well as the methods used to collect the samples or to identify H. pylori. While in some studies biopsies of the lesions were used (7-9), others used swabs (10)(11)(12)(13)(14)(15)(16). Ten out of fifteen studies did not demonstrate a statistically significant association between H. pylori and the presence of RAS (7,8,10,(12)(13)(14)(15)(16)(17)(18). Another important variation that affects the analysis of the studies is the inclusion criteria used to diagnose RAS. As the histopathological features of RAS are nonspecific and the diagnosis is based on clinical grounds, the standardization of patients' selection in future studies is important. There are good reviews about the clinical and diagnostic aspects of the disease (19). The polymerase chain reaction (PCR) method was used to identify the presence of H. pylori DNA in eight studies (8)(9)(10)(11)(13)(14)(15)(16). In two of them, the authors reported a statistically significant association between H.pylori presence and RAS (9,11). While Birek et al. (11) detected H. pylori in 72% of RAS samples using PCR and RT-PCR, Elsheikh & Mahfouz (9) reported that this was mainly observed in lesions localized in mucosa-associ- e189 ated lymphoid tissue of pharynx. However, it is necessary to emphasise that the simple detection of the bacteria in the oral lesion does not mean a causal relationship, as the microorganism may be a "passenger" and may not be the initiating factor of the disease. Most of the studies that employed PCR or nested PCR did not find association between the presence of the bacteria in the oral lesions and its development (8,10,(13)(14)(15)(16). H. pylori DNA was detected between 2% and 38.9% of RAS lesions included in the studies (8,10,(13)(14)(15)(16). None of these studies reported a statistically significant difference be-tween the number of positive samples in the case and control groups. It is interesting that all authors who used the highly sensitive nested PCR method to detect the presence of H. pylori DNA in oral lesions did not find a positive relationship (10,(13)(14)(15) pylori is also a factor that needs to be considered (21). In addition, some of the primers used in the studies could also amplify other Helicobacter species that have also been found in human gut, such as H. fennelliae, H cinaedias (22). Thus appropriate positive and negative controls together with DNA direct sequencing of the PCR product are necessary to define the best PCR conditions and primers that should be used to detect H. pylori in samples collected from the oral cavity.
In two studies, patients with RAS were submitted to endoscopy biopsy to detect H. pylori (23,24). Both studies showed a positive relationship between the presence of the bacteria in the stomach and the occurrence of RAS in the mouth. In the studies of Karaca et al. (23) and Tas et al. (24) 87% and 65% of the patients with RAS, respectively, showed the bacteria in the gastric mucosa.
In four studies, the enzyme-linked immunosorbant assay (ELISA) was used to detect specific antibodies to H. pylori in RAS patients (12,16,17,25). In the study of

Conclusion
The H. pylori can be occasionally detected in RAS lesions and the eradication of the infection may affect the e191 clinical course of RAS lesions by undetermined mechanisms. However, most of the studies do not support the association of RAS ulcers with the presence of the bacteria in the oral cavity and the presence of the bacteria in the ulcer may reflect a passenger infection and not the trigger event. There is no convincing evidence of a direct cause-consequence effect of H. pylori infection and RAS ulcers development. This association requires further investigation by well-design prospective studies.The debate goes on.