Summary

Helicobacter pylori since Marshall and Warren’s discovery has been an object of interest of ga­stroenterologists and many researchers of other specialties. What needs to be highlighted is also the growing interest of dentists in the role of oral residue of H. pylori in oral pathologies such as burning mouth syndrome, periodontitis and gingivitis. With the development of medical techniques more studies using highly specific diagnostic methods are performed in order to determine the transmission pattern of bacterial infection. Suggested faecal-oral and oral-oral routes of bacterial transmission raised interest in molecular biology methods as tools for the study of these environments. Additionally, co-existence of helical and coccoidal forms of H. pylori in the mentioned niches raised the question whether the latter is potentially pathogenic. This is why molecular biology is now giving a great oppor­tunity to explore parts of the human body that could not have been diagnosed before using only gold standard diagnostic methods. Molecular techniques have shown their usefulness in examining the potential virulence of coccoid forms of bacterium. This review was created also to summarize the knowledge about molecular methods, especially different PCR techniques, as diagnostic tools that can help medical teams during regular diagnosis of gastritis.

Key words:Helicobacter pylori review • Helicobacter pylori diagnostics • PCR diagnosis • PCR detection

The use of molecular biology techniquesin H. pylori epidemiological research

H. pylori is a spiral, Gram-negative, microaerophilic bac­terium which colonizes human gastric mucosa and causes severe gastritis and related complications leading to sto­mach and duodenum ulceration as well as – in the long term – to gastric cancer and gastric MALT lymphoma. Since 2005 when two Australian researchers [23] received the Nobel Prize in medicine for discovery of H. pylori and its role as the causative agent in ulcer disease, the num­ber of scientific studies conducted in order to understand the characteristics of H. pylori have increased significan­tly. More extensive epidemiological studies using a high number of patients were performed in order to determi­ne the transmission pattern of bacterial infection. Ways of transmission of H. pylori infection have not been well specified and any attempt to systematize this knowledge has divided the medical community [9]. Epidemiological studies carried out so far enable establishment of relia­ble types of transmission which undergo modifications with the development of medical knowledge. The sugge­sted classification of the types of H. pylori transmission is shown in the table below (Table 1).

Table 1. Types and transmission routes of H. pylori infection

Despite the unsure and hypothetical transmission of H. pylori infection, two routes which have been confirmed by epidemiological studies on large populations of patients seem to be most likely: the transmission of infection thro­ugh the faecal-oral and oral-oral routes [5].

The suggestion that the faecal-oral route of transmission may be of great significance and the easy access to biolo­gical material through which the transmission of infection may occur results in frequent use of faeces as biological material in the epidemiological studies [16]. Many techni­ques are used in order to detect H. pylori in the faeces, e.g. bacterial cultures, enzyme assays and molecular medicine techniques such as polymerase chain reaction (PCR) [5,9].

Due to the abundance and diversity of bacterial flora, faeces as a diagnostic material is problematic as far as obtaining specific results for H. pylori microbial cultures is concerned. From the point of view of detecting H. pylori in the stool, there are two obstacles to the specific determination of H. pylori with the use of microbiological methods. The first methodological problem is related to the high probability of H. pylori occurrence in the coccoid form [29], which makes culture on agar media difficult, and therefore makes the microbiological method demanding and challenging to use in epidemiological studies. The second problem is possible overgrowth of H. pylori by other bacterial strains which in turn may lead to false negative results. Both argu­ments make the establishment of H. pylori microbiological cultures from faeces technically difficult and, subsequen­tly, diagnostically difficult [18,24]. Equally problematic is detection of H. pylori by the PCR method, which, despite the great potential of generic material detection, involves the removal of faecal PCR inhibitors prior to the DNA extraction process [24]. Steps that are recommended in order to remove PCR inhibitors are filtration processes on the polypropylene membranes or chromatographic se­paration on columns [18].

For the process of bacterial DNA amplification to be con­sistent with the current presence of bacterium, and in order to reduce false positive results, which in the case of faeces is particularly important, it is vital to use those fragments of selected genes from bacterial DNA which give the po­ssibility of obtaining a specific result. There are several pre­ferred reactions during DNA amplifications of H. pylori. Among them is the nested PCR method, which proved to be effective during an investigation conducted by Vinette et al. among paediatric patients [39].

Infection through contamination of drinking water by hu­man faeces has been suggested as one of the routes of in­fection transmission. The morphological type that exists in this environment that is harsh for H. pylori has proved to be a coccoidal form of H. pylori [24]. Queralt et al. in their research aimed to confirm the presence of H. pylori in water, in faeces, and in water reservoirs contaminated with human faeces, amplifying successfully the fragments corresponding to the A subunit of the urease gene using nested PCR techniques [29].

The biological fluids used in epidemiological screening are typically excretions and residues of the initial part of the digestive system – saliva, dental plaque and residue from interdental spaces. Considering the suggestions of the im­portance of faecal-oral transmission in the populations of developing countries and oral-oral transmission as the main route of transmission in developed countries [9], it seems appropriate to assume that the reservoir of H. py­lori in the oral cavity may be important during the entire infection process and also during the process of possible reinfection [5,9,32].

Coccoid form of H. pylori – potentially pathogenic?

Observations with the electron microscope conducted by the co-authors of this article, Giovanni Loi and Corrado Serra, showed that the H. pylori helical form converts into a degenerative coccoid form. The process is illustrated by the images taken by the co-authors with the electron mi­croscope which accompany this article. With the use of electron microscopy it was possible to show the process of conversion from the bacillary form of H. pylori to the fully coccoid form (Figure 1). The morphological transformation process begins as a bleb at the bacterial surface (number 1 in the image); the bleb continues to grow and the rod bends, resulting in a U-shaped bacterium (numbers 2-3 in the image); finally, a “ball-like” coccoid form is achieved (numbers 4-6 in the image).

Figure 1. Conversion from the bacillary form of H. pylori to the fully coccoid form. (See description in the text)

This observation is also confirmed by many other scientists who devote their research to exploring the role of H. pylori coccoid form in gastric mucosa infection [11,13,17]. Coc­coid H. pylori forms have been divided into three types: degenerative coccoid form representing dying bacteria of pyknotic structure; a living coccoidal bacterium that can be cultured in agar medium; and viable but non-cultura­ble (VBNC) H. pylori [2,4]. Because of the complexity of the structure and different physico-chemical conditions affecting colonies which form a bacterial biofilm, it has been reported that it is precisely in bacterial biofilms that all forms of coccoid bacterium are present along with the coexisting helical forms. Their presence in vivo may be a result of one of the following conditions. It may be a part of the survival strategy of bacteria, an effect of differing antibiotic concentrations due to different biofilm structures, or due to unequal access of bacterial colonies to nutrients which leads to the differentiation of colonies in this en­vironment [4]. Bacterial ability to transform in the form of grains and a possible return to its helical shape opens new horizons for our understanding of the infection and reinfection transmission with H. pylori [13]. The induction of coccoid form in drinking water reservoirs confirmed by several studies [17], and the presence of H. pylori in marine plankton provide insight into the gravity of the problem of possible infection transmission [11]. In the case of potential reversion from the coccoid to the helical form, there exists a possibility of inducing an active infection on the surface of gastric mucosa [11]; however, in vitro research which attempted to transform coccoid forms of H. pylori into a helical form was unsuccessful in humans. It has therefore been suggested that the transformation of bacteria into the coccoid form reduces its metabolism and induces minor modifications in the physiological processes of the bacte­rium. These changes lead to the inability to recover those bacterial traits which facilitate colonization and infection of gastric mucosa. Observations of H. pylori cultures have shown that the transformation process of the helical form into the coccoid form occurs in the presence of either of two conditions. The first is the occurrence of extremely unfavourable environmental conditions, such as tempera­ture other than 30-37°C, no nutrient medium, gas content level other than the one in the preferred microaerophilic atmosphere, or low concentrations of antibiotics affecting the bacterial culture. In this case the helical form is recon­stituted into a coccoid form. The coexistence of both forms (coccoidal and helical) in various environmental conditions, would explain in this case the survival strategy of bacteria [4,11,17]. Secondly, this transformation may occur during cell degradation due to the end of its life cycle and degen­eration of its virulence factors [40].

As a result of transformation into the coccoid form there occurs bacterial DNA and RNA condensation, but without fundamental changes in the expression of genes respon­sible for virulence of H. pylori, i.e. genes encoding urease, vacuolating cytotoxin and the pathogenic island [40]. For this reason a coccoid form in drinking water and in sea­water tanks accessible by humans would suggest a possible influence on the transmission of infection [4,11,17]. The suspicion that the discussed coccoid form is a survival form in adverse conditions but may give the possibility of trans­mission of infection prompted scientists to test its potential virulence. Wang et al. with the aim of describing the viru­lence of coccoidal forms applied to their research molecular biology methods such as DNA amplification, creation of recombinant plasmids with the use of previously isolated DNA, and subsequent sequencing of obtained genes which were most important for bacterial virulence [40]. In that study, the researchers induced coccoid form formation by exposing the H. pylori NCTC11637 strain to low doses of antibiotics. The conclusion reached by the authors was that sequences of coccoidal and helical forms of H. pylori are 99.7% compatible. It was also observed that the cagA gene which provides pathogenicity of H. pylori was complete in the coccoid form of the bacterium [40].

Speculations about the possible pathogenicity of the coc­coid form led to the search for solutions to this puzzle with the use of an animal model – laboratory mice. In this study bacteria isolated during a biopsy of gastric mucosa of a patient who suffered from gastric ulcers were used to infect the mice. Induction of coccoid forms creation was obtained after the standard microbiological diagnosis of bacterial infection through the stimulation of unfavour­able environmental conditions by dissolving the bacteria in sterile water. The aim of the study was firstly to confirm the presence of cell adhesion factor in H. pylori coccoid form in the mouse gastric mucosa after administrating a suspension of the coccoid form of bacteria; and secondly, to determine whether it is possible to transform the coc­coid form into a helical form in vivo. After the infection procedure of mouse gastric mucosa with coccoidal form of H. pylori was initiated, there was confirmed presence of flagella, an essential element in the process of infection with bacterium of helical morphology. She et al. indicate that the cause of infection may be the bacterial transformation from coccoid form into the spiral form, due to the pres­ence of specific environmental factors, characteristic only for in vivo conditions. It was justified by the presence of spiral H. pylori forms in gastric mucosa biopsy specimens from mice that had been subjected to a solution contain­ing initially only the inducible coccoid form of bacteria. Due to the unconfirmed information about the possibility of inducing the infection through the coccoid form among humans, it was said that the transformation from the form of grains (coccoid form) into the spiral form (helical form) of H. pylori in the human body has to be confirmed and requires further research in order to describe possible pro­cesses which regulate it [31].

Possible applications of molecular methods in detection of coccoidal and spiral H. pylori forms

Studies show a great diversity between different strains of H. pylori infecting one human. That is in agreement with results of an experiment presented by Lukeš et al. [20]. The researchers describe in their paper differences between gastric and oropharyngeal genotypes in viral forms of bac­terium infecting one human. The described experiment was performed using the real-time PCR method based on DNA extracted from saliva and stomach tissue specimens of each patient. The study confirmed that heterogeneity between H. pylori strains can be present also in the level of genes that are most important for bacterium virulence such as cagA and vacA genes in strains involved in infection of one patient. This was also confirmed by Momtaz et al. [25], who claimed to have found different genotypes of H. py­lori in saliva, gastric biopsies and stool of the same patient. The described experiment included testing for presence of ureC and cagA genes and the genotypes of vacA alleles (s1a, s1b, s1c, m1a, m1b and m2) using PCR technique. The conclusions made by Momtaz et al. were that because of high diversity between H. pylori genotypes in saliva, stool and stomach, the latter environment may be most support­ive for growth of H. pylori strains of different genotypes. Genotype diversity in different strains may be caused not only by environmental characteristics, but also by genetic changes or co-infection with another strain.

In 2004 there were more than 23 Helicobacter species. Some of these non-H. pylori species are found in clinical samples, and others colonize animals. What has great importance is that these species have similar genomes. That makes dif­ferentiation with molecular techniques problematic. As described by Suzuki et al., the most closely related species to H. pylori is H. acinonychis, which colonizes the stomach of large felines. Many important virulence factors were also found in H. felis, which even if it lacks cag PAI and vacA, still has the whole ComB system [37]. Additionally, as mentioned before, non-culturable coccoid form of H. pylori is said to be present in both tap water and environmental water. These two facts create a problem with specific spe­cies differentiation. The problem seems not only to be in the detection of coccoid and spiral forms of the bacterium, but also distinguishing Helicobacter from the genotypically similar Campylobacter spp. There are researchers who have set themselves the aim to search for the combination of PCR-based reactions that would provide an effective assay for determining H. pylori either in coccoid or helical form. Shahamat et al. [30] reported in their article that specific species distinction depended on good preparation of the PCR process, based on genetics of H. pylori and the most common species showing similarity, Campylobacter spp. The targets of the Shahamat et al. PCR reaction were the unique region of H. pylori flanking the 16S rRNA gene sequence, urease (ureA), and phosphoglucosamine mutase (glmM) genes. PCR proposed by Shahamat et al. using the 16Sr RNA hypervariable region complete the results ob­tained by PCR amplification with glmM-specific primers, the target of which was the phosphoglucosamine mutase gene. Specific glmM primers allowed detection of Helico­bacter species (pylori and other members of the genus), and did not amplify the following non-Helicobacter species: 4 strains of C. jejuni, and 18 strains of other microbial spe­cies. Amplifications based on the primers specific for the region flanking the 16S rRNA gene of H. pylori discrimi­nate the non-H. pylori species of this genus. Additionally, sequence comparison using the proposed hypervariable region upstream of the 16S rRNA gene of H. pylori can be an easy way to distinguish H. pylori strains from other members of the genus, since this region has sequences that are highly specific for each H. pylori strain.

The molecular method prepared by Nayak et al. [26] was meant to be a valuable source of information about the con­tamination of ground water by viable but non-culturable (VBNC) H. pylori, with a basis that it may be a way of trans­mission of its infection. The reason why scientists prepared a helpful tool to detect a VBNC was to evaluate whether it is possible to use a rapid and effective test of water purity, to avoid waterborne disease transmission. Using an argument that there is a large difference between colonies and species genomes, it should be difficult to evaluate a specific test for detection of H. pylori VBNC form. According to Sisto et al., differences between genomes of coccoid and helical forms of bacterium are not relevant in some conditions. Research­ers have shown that DNA and RNA of the coccoid form are not degraded and that they have expression of the most important virulence and colonization factors such as urease, pathogenic island and vacuolating toxin genes even after a period of 31 days. The only problem was induction of gene vacA, which was not present after 15 days of incubation in most strains apart from strain 190. This, the authors claim, may be due to decrease in mRNA production to below the lower detection limit of the PCR method used in the Sisto el al. research [33]. In other research Can et al. claimed that urease activity was detected in coccoid forms of H. pylori both phenotypically using the rapid urease test and geno­typically by the PCR method [10]. The presence of urease ac­tivity in all coccoid cells induced by different factors showed that transformation from helical to coccoid form influenced urease activity independently from the transforming factor. Determination of an unimpaired ureA region in coccoid cells can confirm that this region of the H. pylori genome is highly protected.

The conclusion can be made based on the presented re­search that coccoid and spiral forms of H. pylori can be specifically detected by molecular biology methods with a mixed procedure of combination of two primers – one based on glmM-specific primers and the second using hy­pervariable region 16Sr RNA, or with primers directed to vacA regions of the bacterium genome [26,30] on the condition that in some cases primers flanking the vacA gene can give false negative results. This was previously described by Nayak et al. as due to prolonged incubation time longer than 16 days.

Differentiation between spiral and coccoidal forms of H. pylori with the PCR test that would specifically answer the question which form is present in the specimen is still problematic. As described in this section there is however a possibility to specifically distinguish both coccoidal and helical forms of H. pylori from other Helicobacter species and closely related bacteria such as Campylobacter. Still the greatest potential for differentiation is the possibility of microbiological culture of the spiral form, and distin­guishing morphological characteristics of the bacterium using specific types of microscopes.

Application of PCRin H. pylori infection diagnostics

Difficulties in biological materials analysis during H. pylori infection diagnostics are due to a long (lasting 6 days) and fastidious growth process of bacterial cultures on agar me­dia. With the development of medical techniques, there ap­peared suggestions to speed up the diagnostics by performing parallel diagnostics using molecular biology techniques. Mi­crobiological diagnosis cannot be abandoned; what is more, in the regular diagnostics it is considered a gold standard on a par with histological diagnostics of gastric mucosa and ure­ase breath test [39]; however, a parallel PCR test can improve the work of medical teams by reducing the results turna­round time, which in turn could enable medical teams to make quicker decisions about patients’ treatment. This is important especially for diagnosis of gastritis in children, in whom heter­ogeneity of distribution of the pathogen was observed. In the case of paediatric diagnosis of H. pylori infection, preparation of biological material and bacterial culture gives the possibility of obtaining a false-negative result [39].

The results of comparative studies testing the techniques used to diagnose H. pylori presence in gastric mucosa bi­opsy specimens from children, such as standard histological techniques, CLO test (Campylobacter-like organism test) and immunological techniques used as tests for antibod­ies to H. pylori performed using ELISA and Western blot methods and techniques of molecular medicine, confirm the usefulness and effectiveness of PCR in analysing the specific presence of infection. Sensitivity of well-construct­ed H. pylori detection reaction can reach even 100%, and its specificity might be greater than any serological tests. Its additional advantage is the fact that it does not require any waiting time for the formation of antibodies [39]. Accord­ing to the recommendations [6,21], the diagnosis of active H. pylori infection should be designed primarily to treat the patient if the result is positive. It is important therefore to select the correct diagnostic methods in order to confirm the presence of bacteria in patients who require antibiotic treatment (it is recommended to first of all diagnose pa­tients with active peptic ulcer disease, patients with peptic ulceration confirmed in their medical history, and patients with gastric MALT lymphoma). What should also be un­derlined is the position of Sugimoto et al., who pointed out the disadvantages of using techniques of molecular medicine when decisions to use the PCR technique as the unique diagnostic method confirming H. pylori infection are made. That is mostly linked with unspecific oligonucle­otides used for bacterium detection in PCR reaction [36].

In patients whose first line eradication treatment has failed, the test for drug resistance of the bacterium should be car­ried out [6,21]. An important diagnostic aid with respect to this problem is molecular medicine methods (PCR) which specifically detect resistance to clarithromycin in biopsy specimens [36]. Because of bacterial resistance to antibiot­ics which according to Malfertheiner et al. in various Eu­ropean countries ranges between 5% and 28% for clarithro­mycin and between 20% and 40% for metronidazole, the possibility of recognizing the initial bacterial resistance to the planned use of antibiotics is usually of key importance for the effective eradication of H. pylori before selection of drugs to be used in the therapy. Resistance to clarithromycin is also a major risk factor when using the treatment recom­mended in Europe, which, besides clarithromycin, consists of amoxicillin and proton pump inhibitors. According to current recommendations presented in the Maastricht III Consensus Report [21], H. pylori resistance to clarithromy­cin test should be performed prior to the treatment if the prevalence of primary resistance is 15-20% [38]. Tankovic et al. in their study show that resistance to clarithromycin is generally caused by two mutations in the 23S ribosomal RNA (rRNA): adenine to guanine transition at position 2142 or 2143, or adenine to cytosine transversion at posi­tion 2142. These two positions participate in the formation of the peptidyl transferase loop of the ribosome. There is a possibility of these mutations’ detection using molecular methods, especially real-time PCR. The technique of as­sessing clarithromycin resistance in biopsy material of gas­tric mucosa by real-time PCR was introduced as a routine in Henri-Mondor Hospital, described by Tankovic et al. in January 2003. The routine histological examination has not been abandoned; however, the microbiological culture was excluded. Microbiological assessment of resistance to drugs was preserved as a basis for the diagnosis in assessing the sen­sitivity of other antibiotics used in the third-line therapy and in case of differences between the results in PCR and his­tological diagnostics, and not for evaluation of resistance to clarithromycin [38]. Moreover, the results of the Chrisholm et al. study showed that H. pylori DNA could be detected by PCR directly in human gastric biopsy samples with high specificity and sensitivity. Chrisholm et al. also developed a PCR assay targeted to the vacA gene – a species-specific and highly conserved locus in H. pylori. This gives a possibility to obtain a valuable result of PCR diagnosis [14].

For most specific primer pairs, the polymerase chain reac­tion method may be used only to confirm the gold stand­ards: culture microbiological tests, histological examination of slices taken during biopsy, or urease tests. Diagnosing an active H. pylori infection only on the basis of a posi­tive PCR result, without the use of the achievements of gastroenterology, microbiology and histology, is not rec­ommended [36].

PCRdetection of H. pylori in material from the oral cavity

Beginning with the discovery of Warren and Marshall [23], H. pylori infection has been in the field of interest of scien­tists from many areas of life science, not only gastroenter­ologists. With the techniques of molecular medicine, it has become possible to examine niches that so far have rendered research problematic due to the lack of diagnostic methods which would be considered as gold standards in diagnos­tics. The reasons for the rising interest in the oral cavity as a potential nesting place of H. pylori which affects the trans­mission process have already been mentioned [15]. It should be noted that the presence of bacteria in the mouth and its relation to infection of the gastric mucosa shows no agree­ment in the scientific community. This may result either from suggestions of co-existence in the mouth of both helical and coccoidal forms, which is recognized by most researchers as a degenerative artefact unable to induce an active infection [40] and whose transformation into a helical form in the human organism has not been confirmed [31], or because of lack of confidence in molecular biology techniques in the di­agnostic assessment of the presence of bacteria in the mouth [36]. Lack of confidence in molecular biology methods has already been pointed out, but the overall assessment of the literature on the subject of the presence of H. pylori in the oral cavity seems to be necessary for a full description of the issue of H. pylori infection.

According to Parrish et al. [27], 325 bacterial strains have been grown so far from dental plaque. However, the devel­opment of techniques for distinguishing all of the actual microbes colonizing the oral cavity is difficult, especially because of the lack of possibility of developing a bacterial culture of some microbes during routine diagnostics [27]. In the explanation of the issue why the gold standard of mi­crobiological culture cannot be used in the oral cavity, the arguments are similar to those presented above, in the section about isolating the bacterial DNA from the stool. H. pylori during microbiological diagnosis of gastritis is problematic because of the long incubation time of the bacterial culture, and the requirement of highly specific substrate and culture conditions. In the case of dental plaque and saliva diagno­sis performance, there is a high possibility of outgrowth by other bacterial colonies whose natural reservoir is located in the mouth, and this process can subsequently affect the growth of H. pylori. Additionally, the presence of H. pylori in the coccoid form inhibits bacterial cultures, which in ef­fect can cause false negative results [15].

It is known that among bacterial flora of the mouth H. pylori is not the only bacteria responsible for urease production. This enzyme, which belongs to the hydrolase class, owes its presence in the mouth to Streptococcus spp., Haemophilus spp., and Actinomyces spp.; hence a positive urease test result in this environment cannot specifically confirm the presence of H. pylori. Taking into account the possibility of receiv­ing a false positive result, this method also does not seem to show any importance in scientific research [15]. The solu­tion to both problems – false-positive urease test results as well as false-negative results of microbiological culture – is to conduct research using molecular techniques, so that the nucleotide sequence comprising a specific gene responsible for encoding the virulence factors of H. pylori will give a spe­cific result [27]. It is particularly important for this reason to use sensitive methods of detection. A highly sensitive test which gives a specific view of the presence of bacteria in the mouth is a well-matched PCR test [15,34].

The use of a one-step PCR test may not be sufficient to carry out the detection of H. pylori in the saliva. The one-step reaction has been evaluated by Mapstone et al. as inad­equate for assessment of the presence of H. pylori in mate­rial from the oral cavity. This is due to a lower degree of H. pylori colonization in the mouth in comparison with the biopsy of the active site of infection in the gastric mucosa.

As far as saliva is concerned, it is required therefore to ap­ply a more specific and sensitive test. Furthermore, Map­stone et al. [22] in their work confirmed this assumption while underlining that the use of a single-step technique is associated with a risk of obtaining false-negative results, despite the use of specific primers [19,34].

Particular attention is paid to the association of the H. py­lori reservoir in the oral cavity with gingivitis and chronic periodontitis [12,35]. It was indeed noted that patients with poor oral hygiene represent a group in which the presence of H. pylori in dental plaque is more likely to be confirmed [1]. It is also suspected that the presence of the bacteria in these two pathologies may lead to oral inflammation and the formation of gingival/periodontal pockets, which develop favourable conditions for bacterial growth [1,35]. Devel­opment of favourable conditions would allow the reinfec­tion of gastric mucosa, particularly in the above-mentioned patients – with poor oral hygiene and periodontal infection – which could foster pathological state development of gas­tric mucosa infection [1]. This statement may be revised by comparing bacteria presence in the oral cavity in patients with good oral hygiene, in which the results of detection of bacteria, although statistically significant, included only 30% of healthy patients [1]. This information suggests the presence of a bacteria reservoir in the dental plaque which plays a role in transmission of H. pylori infection. In addition, researchers show a lot of interest in the ability of H. pylori to adhere to bacteria which cause periodontitis, i.e. Fuso­bacterium spp. [3], and the effect of this co-aggregation on the ability to colonize the mucous membrane, which could initiate the re-infection process [1,38]. One of the studies, conducted by Andersen et al. in order to assess the effec­tiveness of the adhesion of 79 bacterial strains to H. pylori, showed that H. pylori specifically adheres to Fusobacterium nucleatum and Fusobacterium periodonticum of human ori­gin. This process shows the importance of the presence of adhesins on the surface of these two representatives of Fu­sobacterium and their respective receptors on the surface of Helicobacter, which may play a role in the development of periodontal disease as well as the process of reinfection of the gastric mucosa membrane by ingestion of H. pylori [3].

During periodontal disease progression an important factor in the inhibition of disease development is the composition of saliva, as well as its physical conditions (viscosity, proper saliva production of salivary glands) and, which proved to be equally important, a balance between the components of saliva which play an inhibitory role in periodontal disease [28]. It should be noted that the role of proteases in the de­struction of periodontal tissues is of major importance. The presence of protease inhibitors such as salivary leukocyte protease inhibitor (SLPI) has a direct effect on maintaining integrity of the tissue. The protease inhibitors prevent the spread of the disease by neutralizing the action of proteases. Linking stress with increased probability of the occurrence of oral disease, especially in periodontal inflammation in ani­mal and human models, is a subject of interest for research­ers [7]. Stress and psychological care of patients with oral mucosa problems are important not only because of their association with diseases such as periodontitis and gingivitis. Psychiatric disturbances may also be connected with burning mouth syndrome (BMS). A high percentage of patients suf­fering from BMS complain of depression of unknown origin (which results either from chronic mouth pain or otherwise is not connected with any burning symptoms).

In the case of patients with symptoms of burning in the oral cavity and clinically healthy oral mucosa detailed medical history should reveal systemic factors which might result in burning symptoms. There exists a strong link between those patients and patients who complained of oral burning before treatment for H. pylori. As Brailo et al. confirmed in their research, “true” burning mouth syndrome diagnosis should be established when all other systemic and local reasons for burning are excluded. Unfortunately, as Brailo continues, the “true” burning mouth syndrome still shows limited therapeutic options [8]. Particular interest should be paid to infection with H. pylori due to the high percentage (79%) of patients whose burning symptoms were resolved after H. pylori eradication treatment. Additionally, in patients with burning symptoms clinical examination of the oral cavity – candida swab, oral galvanism measurement, salivary flow rate and haematological screening (with specific attention to serum ferritin, blood glu­cose levels and H. pylori antibodies) – should be performed in order to identify possible underlying disturbances. Brailo et al. also suggested that due to the significantly higher prevalence of gastritis in patients with BMS together with the reported incidence of 3.2 times more gastrointestinal symptoms in BMS patients, each patient with burning mouth syndrome should consult a gastroenterologist [8].

Acknowledgments

The linguistic and editorial assistance of Irena Wypych is gratefully acknowledged.

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The authors have no potential conflicts of interest to declare.

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