Isolation of viable Helicobacter pylori in the tonsillar tissues of chronic tonsillitis patients

This item has no abstract available. Use the links below to access the fulll text. Background: Helicobacter pylori is a Gram-negative, spiral, microaerophilic bacterium that originally colonizes the human stomach. H. pylori was reported also present in the mouths of both gastric biopsy-positive and gastric biopsy-negative patients, tonsil and adenoid tissues, and middle ear. The objective of this work is to isolate the viable H. pylori which may colonized the tonsillar tissue of chronic tonsillitis patients. Methodology : A total of 19 patients suffering from chronic tonsillitis which were admitted for elective tonsillectomy were included in the study. Specimens of tonsilar tissue were cultured for H. pylori . To confirm the presence of H. pylori , histological examinations were performed using modified Giemsa staining and immunohistochemistry assay . Results: Viable H. pylori were isolated in tonsilar tissue of 3 out of 19 patients (15.7%) suffering from chronic tonsillitis. The result was confirmed using modified Giemsa staining and immunohistochemistry assay. Conclusion: Viable H. pylori colonization in tonsilar tissue of chronic tonsilitis patients may be detected by combination of culture and histological examinations.

Helicobacter pylori is a Gram-negative, spiral, microaerophilic bacterium that originally colonizes the human stomach.Chronic H. pylori infection has been associated with chronic gastritis, peptic ulcer disease, atrophic gastritis, mucosa associated lymphoid tissue (MALT) lymphoma, and gastric cancer [1].H. pylori has been also detected in the mouth and the middle ear as well as in tonsillar and adenoid tissues [2][3][4][5].However, the relationship of H. pylori with the pathogenesis of diseases that involve these organs is controversial.
Accumulated reports show controversies regarding the role of H. pylori in oropharyngeal infection.The hypothesis that H. pylori can colonize tonsillar and adenoid tissue has not yet been well elucidated due to the difficulty in isolating viable H. pylori [3,6,7].The results of this study indicate that viable H. pylori can be detected in the tonsillar tissues of chronic tonsillitis patients using a combination of conventional culture and histological examination employing modified Giemsa staining and immunohistochemistry.
The study included nineteen patients with chronic tonsillitis who were admitted for elective tonsillectomy.Patients were evaluated as having chronic tonsillitis based on Ballantyne and Groves criteria [8] .All patients underwent tonsillectomy under general anesthesia.The tonsillar tissues were divided in two parts and subsequently used for microbiological and histological analysis.Informed consent was obtained from all patients or their parents.
Specimens obtained from patients were directly inoculated into Stuart transport medium.Soon after arriving at the laboratory, specimens were inoculated directly on Trypton Soy Agar (TSA) medium (Oxoid Ltd, Cambridge, UK) supplemented with 5% defibrinated sheep blood and H. pylori selective Skirrow's medium supplemented with vancomycin, trimethoprim, polymyxin B, and amphotericin B (Sigma-Aldrich, St. Louis, USA).
Plates were incubated micro-aerobically using the gas generating kit (Mitsubishi Gas Chemical Co Inc, Tokyo, Japan) at 37°C for 5 to 7 days.Colonies of suspected H. pylori were identified by Gram and Giemsa staining, and a positive reaction of the urease test.
Tonsillar tissue samples were fixed in 10% (v/v) formalin, then embedded in paraffin, and subsequently used for modified Giemsa staining and immunohistochemistry. Modified Giemsa staining was performed according to the standard procedure described elsewhere [9].Immunohistochemistry detection was performed using a specific H. pylori antibody (Dako, Glostrup, Denmark) Briefly, tissue sections were immersed in 10 mmol/L sodium citrate buffer, pH 6.0, and then autoclaved at 120°C for 10 minutes.The sections were then treated with 3% H 2 O 2 for 10 minutes at room temperature to inactivate endogenous peroxidase, and then they were blocked with 10% goat serum for 20 minutes at room temperature.The sections were incubated with a-H.pylori Ab (Dako, Glostrup, Denmark) overnight at 4°C.The sections were washed with PBS and incubated for 20 minutes with biotinylated goat antirabbit IgG (Dako, Glostrup, Denmark).The sections Viable H. pylori was detected by conventional culture in three male patients out of 19 patients (15.7%) and confirmed by histology using modified Giemsa staining and immunohistochemistry (Figure 1).Culture results were in 100% agreement with modified Giemsa staining and immunohistochemistry results.
Culture is the gold standard method for the determination of H. pylori.It indicates the presence of viable bacteria, though several factors may lower its sensitivity, such as antibiotic administration, local disinfection, and anesthetic application.Modified Giemsa staining and immunohistochemistry staining were used to confirm culture results due to their superior and reproducible capability to detect H. pylori in tissue samples.
Some studies have suggested that H. pylori may have an association with oropharyngeal infection [3,4,7].However, this notion was not supported by the work of several groups which were not able to detect the colonization of H. pylori in tonsillar tissue [6,10].These contradictory results may be due to the specific characteristics of each study population or to the variety of methods employed in the studies.Yilmaz et al. [5] were the first who isolated H. pylori from tonsillar tissue cultures from patients suffering from otitis media with effusion.Our study appears to be the first reporting isolation of viable H. pylori from the tonsillar tissues of patients with chronic tonsilitis.Eyigor et al. [11] reported that H. pylori can be detected in tonsillar tissue of 5.5% of chronic tonsillitis patient by rapid urease test (RUT) but not with PCR.Vayisoglu et al. [12] detected H. pylori using RUT in only 2.2% of adenoidectomy specimens and in none of the tonsillectomy specimens.A positive result was not obtained in any tonsillectomy specimen using immunohistochemistry.Our results showed H. pylori colonization in the tonsillar tissue of 15.7% of patients suffering from chronic tonsillitis.The different findings may be related to distinct population characteristics.
The combination of culture and histological examination may be useful in detecting H. pylori colonization of tonsillar tissue in chronic tonsillitis cases.

Figure 1a .
Figure 1a.Giemsa staining of H. pylori colonies cultured on selective medium

Figure 1c .
Figure 1c.Detection of H. pylori using immunohistochemistry