The role of adenotonsillar tissues as a reservoir for Helicobacter pylori and Helicobacter hepaticus.

AIM: The aim of current study is to investigate whether tonsillar and/or adenoid tissue of patients with chronic adenotonsillitis plays a reservoir role for Helicobacter pylori (H. pylori) or Helicobacter hepaticus (H. hepaticus).
BACKGROUND: Recently, there have been arguments ragarding Helicobacter pylori (H. pylori) being reserved in adenotonsillar tissue. PATIENTS AND METHODS: This study was performed with 90 patients with the diagnosis of chronic tonsillitis and adenoid hypertrophy, mean age 36 ± 22, 32 (36%) female and 58(64%) male. Presence of H. pylori and H.hepaticus were detected by glmM gene and 16S rRNA specific primers respectively.
RESULTS: Of all patients 58 (65%) were found seropositive for H. pylori IgG while only 7(8%) patients had gentile gastrointestinal (GI) symptom, all gastritis. H. pylori and H.hepaticus was not detected in any of the patients by PCR.
CONCLUSION: There was no correlation between GI symptom and/or seropositivity of H. pylori with presence of H. pylori and H. hepaticus in adenotonsillar tissues. Our results did not support the role of adenotonsills as a reservoir for H. pylori or H. hepaticus.

Introduction

It is well documented that Helicobacter pylori (H. pylori) infection is worldwide, however, the precise transmission way have not been fully determined and remains vague. Transmission via the fecal-oral and oral – oral transmission is believed as the major mechanism of H. pylori distribution (1–3). H. pylori has been detected in the oral cavity samples from supragingival plaque, dental plaque and saliva by polymerase chain reaction (3–7), but culture of the bacteria from oral cavity samples has been very rare. More recently, it has also been suggested that adenotonsillar tissues may act as a reservoir for H. pylori, thereby several studies evaluated the presence of H. pylori in adenoid and tonsil specimens (8, 9). There is a hypothesis that tonsillectomy may provide protection against H. pylori infection of the stomach since the tonsils may have a role as reservoirs for recurrent H. pylori infections (10). If infection of tonsillar tissue does indeed have an impact on H. pylori colonization of the gastric mucosa, clinicians would benefit from having access to an easy, sensitive, and specific method of verifying the presence of H. pylori in tonsillar tissue. Eradicating known H. pylori from tonsillar tissue by adenotonsillectomy might make it easier to eradicate it in gastric mucosa.

In addition to H. pylori, several non-pylori helicobacter species such as Helicobacter heilmannii, Helicobacter felis and Helicobacter bizozzeronii have been detected in the human stomach while some others like Helicobacter hepaticus and Helicobacter bilis, have been shown in the human enterohepatic tracts (11–16). The original source of non-pylori Helicobacter species has been reported to be animals including dogs, cats, mice and pigs (13–16) in which they have in some cases been associated with human disease such as gastritis, gastric ulcers, MALT lymphomas and extra gastrointestinal diseases (13, 15–20). These non-pylori Helicobacter species can be transmitted to humans via close contact and have in some cases been associated with pathology. However the exact route of transmission and possible role of adenotonsillar area as a reservoir of these bacteria remains unknown.

In the current study, we aimed to investigate whether adenoid and/or tonsillar tissue from patients diagnosed with chronic adenotonsillitis was a reservoir for H. pylori and/or H. hepaticus using molecular techniques. Comparison of the surface versus the core of adenotonsillar tissue was investigated to determine contamination with H. pylori and H. hepaticus. Informed consent was obtained from all patients, and the protocol was approved by the ethical committee of Research Center for Gastroenterology and Liver Diseases in Shaheed Beheshti University of Medical Science.

Patients and Methods

Out of 120 patients who underwent adenotonsillectomy in Taleghani General Hospital between April 2009 to February 2010, 90 patients were included in the study. The criteria for undergoing tonsillectomy was six or more tonsillitis attacks in a year, while the criteria for adenoidectomy was defined as 2/3 blockage of the choana on endoscopy which resulted in obstruction symptoms. All patients underwent adenoidectomy and/or tonsillectomy operations under general anesthesia. In the operating theatre, following excision of the tonsils or adenoids all surgical specimens were washed with sterile normal saline and were cut into the desired sections using a newly sterilized blade under sterile conditions. Three core and three surface samples (overall six with 2-3 mm wide) were obtained from each adenotonsillectomy patients (one from each tonsil and one from the adenoid). Following the preparation of the samples they were immediately sent to microbiology laboratory in sterile screwcap tubes containing normal saline or TE buffer

The H. pylori seroprevalence in selected patients was measured by enzyme-linked immunosorbent assay (ELISA) for the immunoglobulin G (IgG) antibody against H. pylori (Pishtaz Teb, Tehran, Iran).

DNA from each H. pylori isolate was extracted using a commercially available kit (Qiagen, Hilden, Germany). The glmM (ureC) gene was used for molecular detection of H. pylori strains. The primer sequence of glmM used in current study was as follows: forward 5-GCT TAC TTT CTA ACA CTA ACG CGC-3 and reveres, 5-GGA TAA GCT TTT AGG GGT GTT AGG GG-3. Helicobacter hepaticus specific primers; forward: 5′- GAAACTGTTACTCTG -3′; reverse: 5′- TCAAGCTCCCCGAAGGG -3′ were used to amplify a 405 bp fragment of the 16S rRNA gene (19, 21). Primers PC04 (5′-CAA CTT CAT CCA CGT TCA CC-3′) and GH20 (5′-GAA GAG CCA AGG ACA GGT AC-3′) were used to amplify a 268-bp fragment of the human β-globin gene as a control gene to monitor specimen processing and DNA extraction (22). All PCR mixtures were prepared in a volume of 25 µL containing 1 × PCR buffer, 500 nM of each primer, 1.5 mM MgCl2; 200µM each dNTP, 1.5U Taq DNA polymerase, and 300 ng DNA sample. The mixtures were placed in a thermocycler (Eppendorf AG 22331, Hamburg, Germany), PCR products were visualized by electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and examined under UV illumination. The primers and methods for H.pylori detection were controlled by using same primers, material and methods from gastric biopsies.

The Fisher's exact test and the Chi-square test were used for analysis of categorical data. Analysis was done using Sigma Stat for Windows V2.03 (SPSS, Chicago, IL). A p-value of <0.05 was considered statistically significant.

Results

All 90 patients underwent adenotonsillectomy included in current study. The age of the patients were 36 ± 22 varied from 6 to 80. Of patients 32 were female (36%) while 58 were male (64%). All patients have not received any antibiotic at least one month prior to adenotonsillectomy. None of the symptoms and physical examination findings supported the existence of any preoperative gastroesophageal reflux disease (GERD). Serology for H. pylori IgG showed that 58 (65%) of 90 patients were positive in ELISA test regardless to clinical manifestation. Although by increasing age, we detected an increased prevalence of H. pylori, however there was no statically significant correlation between patients’ age and seopositivity of H. pylori (p > 0 .05) in our study. Overall 7(8%) patients had gastrointestinal disorder; all gastritis with no sign of ulcer or cancer, mean age 45± 20. We could not find any correlation between GI symptom and other variable such as age and sex. Successful DNA extraction was confirmed by positive PCR product for the human β-globin gene as an internal control in all of the specimens. However, no H. pylori gene was detected in any of the specimens, both in the core and surface of samples. Also, once we performed PCR for Helicobacter hepaticus as a member of extra gastrointestinal Helicobacter spp. No samples were found positive for DNA in all studied specimens.

Discussion

Helicobacter pylori is a major human pathogen that is estimated to infect half of the worlds’ population and more than 90% of the Iranian people (5). H. pylori infection is a major cause of chronic gastritis, peptic ulcer disease (PUD), gastric carcinoma (GC) and mucosa associated lymphoid tissue (MALT) lymphoma (23, 24). Although the stomach is believed to be the primary reservoir for H. pylori, it has been detected from dental plaque, the oral cavity and saliva (4). Thus, it is possible that the upper aero-digestive tract including tonsil and adenoid tissue might act as a reservoir for H. pylori especially for oral-to-oral mode of transmission. This idea is based on the results of previous studies, in which the bacteria were detected in dental plaques, oral lesions and saliva (4, 25, 26). Determination of H. pylori colonization can be made either invasively using culture of biopsy specimens collected at endoscopy or noninvasively using serologic analysis, the rapid urease test (RUT), CLO-test (commercially prepared RUT) or molecular techniques such as PCR (polymerase chain reaction). Of advantage of PCR, that it is able to detect very low numbers of microorganisms, including the non- cultivable coccoid form of H. pylori. Also if highly specific primers are used, PCR does not cross react with other urease producing organisms as has been observed using RUT. Thereby PCR is one of the best choices for detection of H. pylori and other microorganisms of interest based on their specific genes targets. The results of the current study would suggest that adenotonsillar tissue is not a reservoir for H. pylori. This finding is consistent with previous studies which failed to detect H. pylori colonization of adenotonsillar samples (27–30). In contrast, a number of studies have reported the detection of a high prevalence of H. pylori in adenotonsillar tissues (9, 31, 32). Of reasons may account for the divergent results in detection of H. pylori from adenotonsillar specimen in different studies, might be the type of H. pylori detection method as main issue for such diversity. Examination of previous studies shows that in the majority of those in which H. pylori was detected in inadenotonsillar tissues, the RUT (rapid urease test) was employed (9, 32). Given the abundance of bacteria in the non- acidic oral cavity the UBT is likely to lead to a high rate of false-positive results due to the known presence of other urease producing bacteria (30, 33, 34). Thus the high rates of positivity in the studies conducted using only RUT test are likely to be unreliable. In the current study, the primers, materials and methods were controlled as by using same material and methods we were able to detect H. pylori from gastric biopsy sources (35–37), Therefore, we think that the negative results for H. pylori, observed in this study, are not caused by the used material or techniques.

Recently increasing evidence that non-pylori helicobacters may play a role in human gastrointestinal and extra gastrointestinal disease has been reported. To date, several studies have investigated the adenotonsillar tract as reservoir of H. pylori, while none have examined these tissues as possible reservoirs for non-pylori helicobacter isolates. Thereby we considered H. hepaticus in our study program. Similar to H.pylori results, all samples were negative for H. hepaticus too, which shows that this bacteria like to H.pylori do not colonize on adenotonsillar tissue surface or core. As our knowledge, this is the first report regard to study of non-pylori helicobacter strain in adenoid and tonsils tissues. In conclusion, the fail of current study to detect any positive PCR result for H. pylori DNA is in agreement with majority of studies which have excluded the possible role of tonsils and adenoid as reservoir of H.pylori infection. Also we could not detect any H. hepaticus colonized on adenotonsillar tissues which result out the possible role of adeoid and tonsils as a reservoir for this bacterium.