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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 20  |  Issue : 3  |  Page : 273-279

Detection of Helicobacter pylori in paranasal sinus specimens from chronic rhinosinusitis patients


1 Department of Otorhinolaryngology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
2 Department of Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

Date of Submission02-Jan-2022
Date of Decision08-Mar-2022
Date of Acceptance17-Mar-2022
Date of Web Publication11-Oct-2022

Correspondence Address:
MD Mohamed K Al Awady
Assistant prof of Otorhinolaryngology, Faculty of Medicine, Alazhar University Al-Moktom, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/azmj.azmj_2_22

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  Abstract 


Background and aim Chronic rhinosinusitis (CRS) is an upper respiratory-tract disease, which affects general health of patients. This study aims to detect Helicobacter pylori in paranasal sinus specimens of patients having CRS and to approve if there is a relation between H. pylori and CRS.
Patients and methods This study included 60 patients complaining of nasal and gastroesophageal reflux disease (GERD) symptoms. According to computed tomography findings, patients were divided into group A: this included 30 patients, they had chronic sinusitis, or nasal polyp and GERD symptoms, and group B: this included 30 patients, they had inferior-turbinate hypertrophy without sinus affection and with GERD symptoms. All patients were submitted to endoscopic surgery, under general anesthesia, and the samples of diseased sinus mucosa or nasal polyp in group A and the samples of inferior turbinate in group B were sent for histopathological and microbiological examination.
Results In group A, rapid urease test (RUT) was positive in 10 (33.3%) patients and negative in 20 (66.6%) patients, while immunohistochemistry (IHC) was positive in five (16.5%) patients and negative in 25 (83.5%) patients. In group B, RUT was positive in five (16.5%) patients and negative in 25 (83.5%) patients, while IHC was positive in 0 (0%) patients and negative in 30 (100%) patients.
There was a significant difference between RUT and IHC results in both groups (P=0.032).
Conclusion H. pylori infection has possible role in chronic sinusitis. More studies are required to confirm this correlation.

Keywords: chronic rhinosinusitis, Helicobacter pylori infection, immunohistochemistry


How to cite this article:
Al Awady MK, Hamed Alahmer MM, El-Morsy MA, Mostafa OM. Detection of Helicobacter pylori in paranasal sinus specimens from chronic rhinosinusitis patients. Al-Azhar Assiut Med J 2022;20:273-9

How to cite this URL:
Al Awady MK, Hamed Alahmer MM, El-Morsy MA, Mostafa OM. Detection of Helicobacter pylori in paranasal sinus specimens from chronic rhinosinusitis patients. Al-Azhar Assiut Med J [serial online] 2022 [cited 2023 Jan 27];20:273-9. Available from: http://www.azmj.eg.net/text.asp?2022/20/3/273/358038




  Introduction Top


Chronic rhinosinusitis (CRS) is an upper respiratory-tract disease, which affects general health of patients [1]. CRS has different causes such as allergy, infections, and inflammatory reactions [2],[3]. Helicobacter pylori has potential role in gastroesophageal reflux, so, it can cause supraesophageal mucosa injury and upper respiratory-tract inflammation [4],[5].

Also, there is a relationship between H. pylori in some diseases such as cardiovascular, respiratory disorders, and head and neck malignancies [6],[7]. H. pylori has been detected in oral ulcers, and in tonsillar and adenoidal tissue [8],[9]. Also, it has been discovered in sinus mucosa of CRS [10],[11] in later studies, according to the size of different samples and methods of detection [12],[13].

Recently, several studies proved that gastroesophageal reflux has potential role in CRS. If the association between chronic sinusitis and H. pylori is confirmed, it may give great effect in the treatment of chronic sinusitis and may prevent recurrence [14].

This study aims to detect H. pylori in paranasal sinus specimens of patients having CRS and to approve if there is a relation between chronic sinusitis and H. pylori infection.


  Patients and methods Top


This study was done at Al-Azhar University Hospitals, at the time from May 2020 to August 2021. This included 60 patients complaining of manifestations of rhinosinusitis, which were discharge, nasal obstruction, headache, and facial pain, that were not responding to medical treatment.

This study is approved by Ethical Committee of Faculty of Medicine Al-Azhar University.

Informed written consent was obtained from all patients submitted to this study. The study is conducted in accordance with Helsinki standards as revised in 2013.

Inclusion criteria:
  1. Patients had chronic sinusitis, due to either maxillary/ethmoid or pan-sinusitis.
  2. Patients with inferior nasal-turbinate hypertrophy.
  3. Patients had usual manifestation of gastroesophageal reflux disease (GERD) (heartburn, acid taste, and regurgitation).


Exclusion criteria:
  1. Patients had nasal mass other than nasal polypi such as nasal granulomas (e.g. rhinoscleroma or Wagner granuloma), benign tumors of the nose, paranasal sinus carcinoma, or patients had fungal sinusitis.
  2. Patients had no symptoms of GERD (heartburn, regurgitation, and acid taste).


According to computed tomography (CT) findings, all patients were divided into two groups:

Group A: including 30 patients, they had chronic sinusitis or nasal polyp and GERD symptoms (heartburn, regurgitation, and acid taste).

Group B: including 30 patients, they had inferior-turbinate hypertrophy (ITH) without sinus affection and with GERD symptoms.

All patients were asked about the usual manifestation of GERD (heartburn, acid taste, and regurgitation), as well as whether they had used medical treatment such as histamine (H2)-receptor blockers or antibiotics and antacids, before surgery.

All patients were asked about the main complaint of rhinosinusitis such as either postnasal discharge, nasal obstruction, headache, facial pain, chronic cough, and temporary headache, and otorhinolaryngological examinations were done for all patients.

Before surgery, serologic tests were done as for all patients, enzyme-linked immunosorbent assay tests were used to detect specific H. pylori antibodies [immunoglobulin G (IgG)] in all cases, and fresh stool samples were used to examine H. pylori antigen in both groups to confirm gastric condition.

CT scan on the nose and paranasal sinuses were as done to detect the type of nasal pathology and any sinus affected.

All patients were submitted to endoscopic surgery, under general anesthesia, and the sample of resected polyp or of diseased sinus mucosa in group A and the sample of inferior turbinate in group B were sent for histopathological and microbiological examination.

Sample processing

Tissue samples were obtained from sinus mucosa or from inferior nasal turbinate during surgery, without contamination, and all samples were collected and put into the paraffin block for histopathological examination and bacteriological study.

Histopathological study

Fixation of samples in 10% buffered formalin, then processing was done for paraffin embedding. Hematoxylin and eosin stain was important for diagnosis of tissue affection, detection of the bacteria, and any changes in the size and shape of cells associated with H. pylori infection.

For cytology and tissue culture, fresh tissue was stained with 2% modified Giemsa solution done to produce air-dried touch smears. H. pylori appears in examination under ×100 objective as gray–blue ‘S’-shaped bacteria ([Figure 1]).
Figure 1 Gram-stained film for Helicobacter pylori from culture. Original magnification ×1000.

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Immunohistochemistry

Immunohistochemistry (IHC) is an accurate and sensitive method to detect H. pylori. Deparaffinzed 3–4-μm-thick sections from the tissue blocks were brought to water and incubated with anti-H. pylori polyclonal antibody.

Staining was performed with peroxidase-conjugated streptavidin. Color was revealed by incubation with DAB solution, followed by washing and counterstaining with hematoxylin. Immune reaction was defined as positive, when immune-reactive structures were detected on the surface of the epithelium, and/or on the surface of nasal and sinus glands ([Figure 2]).
Figure 2 Immunohistochemistry and immunostained Helicobacter pylori organism (curved spirochete-like organism) (arrows) are seen distributed in glands of chronic sinusitis. Magnification ×1000.

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Bacteriological study

Tissue culture and rapid urease test (RUT) was based on urease enzyme secreted by H. pylori, which converts urea into ammonia and bicarbonate. The samples were collected and placed immediately into the bottle of urease test and examined the color change, color change to bright pink (fuchsia) is considered positive urease test ([Figure 3]).
Figure 3 Rapid urease test of Helicobacter pylori shows color change to bright pink (positive urease test).

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Media for Helicobacter pylori detection

Medium for transportation:
  1. In all, 1 ml of sterile physiological saline in bottles put in ice bags.
  2. Media for isolation of H. pylori:


Dent’s medium that is formed of:
  1. Blood agar base that has the following formula/l:
    1. Protease peptone: 15.0 g.
    2. Liver digest: 2.5 g.
    3. Yeast extracts: 5.0 g.
    4. Sodium chloride: 5.0 g.
    5. Agar: 12.0 g.
    6. pH 7.4±0.2.
  2. About 5–7% defibrinated horse blood (oxoid CM271).
  3. H. pylori-selective supplement consists of:
    1. Vancomycin: 5.0 mg.
    2. Trimethoprim lactate: 2.5 mg.
    3. Cefsolodin: 2.5 mg.


Then, 2 ml of sterile distilled water was added and mixed gently.
  1. Media for identification of isolated organisms:
    1. Medium for RUT:


It was prepared as follows:
  1. Sodium chloride: 0.5 g.
  2. K2HPO4: 0.2 g.
  3. Distilled water: 99.0 ml.
  4. Urea 40%: 6.0 ml.
  5. Phenol red 0.4%: 1.0 ml.


These ingredients were mixed together and stored in a refrigerator in small plastic tubes at −4°C ([Figure 3]).

Statistical analysis

Recorded data were analyzed using the Statistical Package for Social Sciences, version 20.0 (SPSS Inc., Chicago, Illinois, USA). Quantitative data were expressed as mean±SD and range. Qualitative data were expressed as frequency and percentage. The following tests were done:

Paired-sample t test of significance was used when comparing between related samples.

χ2 test of significance was used in order to compare proportions between qualitative parameters.

The confidence interval was set to 95% and the margin of error accepted was set to 5%. P value was considered significant when it was less than 0.05.


  Results Top


In the study group, 30 patients with nasal polyposis or CRS, including 25 (82.5%) males and five (16.5%) females, underwent endoscopic sinus surgery. In the control group, there were 30 patients with ITH, including 18 (59.4%) males and 12 (39.6%) females who were submitted to endoscopic surgery. In the study group, the age of patients was 20–70 years, while in the control group, the age range of patients was 18–60 years, the mean age of the study group was 39.6±8.7 years, while the mean age of the control group was 29.7±7.2 years. There was no significant difference in age or sex between both groups (P>0.05).

According to CT findings, we observed that the patients of the study group who had maxillary/ethmoid sinusitis ([Figure 4]) were 15 (50%), five (16.5%) patients had maxillary sinusitis, and 10 (33.3%) patients had pan-sinonasal polyposis ([Figure 5]).
Figure 4 CT paranasal sinus coronal cut shows maxillary and ethmoid sinusitis. CT, computed tomography.

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Figure 5 CT paranasal sinus coronal cut shows panasinonasal polyposis. CT, computed tomography

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While in the control group that included 30 patients who had bilateral ITH, seven (23.1%) patients of them had concha bullosa, while 18 (59.4%) patients of them had deviated nasal septum ([Table 1]).
Table 1 Computed tomography findings in the study and control groups and their percent

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Serologic results

H. pylori– immunoglobulin M antibodies positively were positive in 62.7% (19/30) of case groups and positive in 26.2% (8/30) of control group with significant difference (P=0.027; χ2=3.147).

H. pylori (IgG) antibodies were significantly higher in the patient group due to previous immunity (27/30) 89.1% versus (16/30) 52.5% of controls (P=0.013; χ2=5.223) ([Table 2]).
Table 2 Serologic results in both groups and their percent

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Histopathological and immunohistochemical results

IHC is accurate and sensitive for H. pylori detection. By IHC, we can identify low counts of bacteria and fragmented bacteria. IHC was positive in five (16.5%) cases of the study group, while it was positive in 0 (0%) cases of the control group with significant difference (P=0.04; χ2=6.926).

Hematoxylin and eosin E-stained sections showed H. pylori on the surface of the epithelium, while H. pylori appears as curved S-shaped gray–blue bacterium in modified Giemsa-stained smears ([Table 3]).
Table 3 Comparison between study and control groups regarding immunohistochemical analysis

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Comparison between the results of IHC and RUT in study and control groups.

IHC was more accurate and sensitive than RUT performed in the study. In the study group, RUT was positive in 10 (33.3%) patients and negative in 20 (66.6%) patients, while IHC was positive in five (16.5%) patients and negative in 25 (83.5%) patients.

In the control group, RUT was positive in five (16.5%) patients and negative in 25 (83.5%) patients, while IHC was positive in 0 (0%) patients and negative in 30 (100%) patients. There was a significant difference between patient and control groups as regards RUT and IHC-positive results (P=0.032) ([Table 4]).
Table 4 Comparison between results of immunohistochemistry and rapid urease test in study and control groups

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  Discussions Top


CRS is a chronic disease that affects general health. There is a relation between H. pylori infection or GERD and pathogenesis of CRS in some patients. Several studies have proved high incidence of GERD in CRS patients [15].

Park et al. [16] proved that H. pylori was more presented in CRS patients than in controls. GERD transmits HP from the stomach to the nasal cavity, so that all patients in this study were complaining of manifestations of rhinosinusitis and GERD symptoms (heartburn, regurgitation, and acid taste) [16].

Recent studies proved that manifestation of sinusitis has improved by medical treatment of GERD with antibiotics in 79% of patients. Also, identification of H. pylori in the respiratory system suggests an association between H. pylori and CRS [17].

In this study, we found that H. pylori–immunoglobulin M positivity was significantly different between patient and control groups, and also, H. pylori (IgG) was significantly higher in patient group due to immunity from previous infection.

This was similar to Noorbakhsh et al. [18], who found that H. pylori (IgG) was positive, 71% (44/62), in patient group and was positive, 32% (8/25), in the control group with a significant difference (P=0.001), but there was no significant difference in H. pylori–IgA antibodies, in his study, it was 14.5% (9/62) in cases and was 4% (1/25) in controls (P=0.27, odds ratio=4.1).

H. pylori antibody (enzyme-linked immunosorbent assay) tests are less accurate than PCR for diagnosis of CRS caused by H. pylori. Serology confirms active CRS caused by H. pylori and excludes simple colonization. Therefore, positive serological tests alone are not enough for diagnosis of active H. pylori infection causing CRS [19].

IHC is more accurate and sensitive than RUT for H. pylori detection. By IHC, we can identify low-count bacteria or fragmented form of bacteria [19].

In this study, in the patient group, RUT was positive in 10 (33.3%) patients and negative in 20 (66.6%) patients, while IHC was positive in five (16.5%) patients and negative in 25 (83.5%) patients. There was a significant difference between patient and control groups as regards RUT and IHC-positive results (P=0.032).

The results of this study agree with Ozdek et al. [11], who detected H. pylori by IHC analysis in 18% of the nasal and maxillary sinus tissue from patients with CRS.

Also, Ozdek et al. [11] in their study showed significant differences in PCR and IHC results when compared with RUT results. This was similar to Kim et al. [12], who found that RUT was positive in 39.6% in the study group and was 6.9% in the control group. IHC was positive in 41.7% in the study group and was 27.6% in the control group. They concluded that there is significant difference between study and control groups (P<0.05) as regards RUT and IHC.

The degenerated forms or low concentration of H. pylori may lead to weakly positive or false-negative urease test and culture, this matches with Szczygielski et al. [19] and Nemati et al. [20] by urease test, who could not confirm the association between H. pylori and CRS. Kaviani et al. [21], reported false positivity in their study due to urease activity of some other bacteria, also, they concluded that urease test is not an accurate method for detection of H. pylori, it has to be confirmed through IHC or PCR.

Conclusion

H. pylori was detected in diseased sinus mucosa. This correlation approves that H. pylori has a role in pathogenesis of CRS, however, more studies are needed to confirm this correlation. We recommend IHC as the best method for H. pylori infection.

We recommend addition of antireflux and anti-H. pylori regimen to the treatment protocol of chronic sinusitis patients, especially in resistant cases associated with GERD symptoms.

Recommendation

We recommend addition of antireflux drugs and anti-H. pylori regimen to the treatment of chronic sinusitis patients, especially if associated with GERD symptoms.

Also, we recommend that more studies are required to confirm the correlation between CRS and H. pylori, using a larger number of patients in multicenters of laboratory investigations concerned by different methods for H. pylori detection.

Acknowledgements

Limitations of this study: this study has some limitations of being small number of patients, and laboratory investigations were done in a single-place center.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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