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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 16  |  Issue : 3  |  Page : 270-274

Detection of pepsinogen and Helicobacter pylori in preschool age children with secretory otitis media


1 Department of Otorhinolaryngology, Faculty of Medicine, Al-Azhar University, New Dameitta, Egypt
2 Department of Microbiology, Faculty of Medicine, Al-Azhar University, New Dameitta, Egypt

Date of Submission05-Aug-2018
Date of Acceptance27-Jan-2019
Date of Web Publication15-Apr-2019

Correspondence Address:
Wael F Ismaeil
New Damietta City, New Dameitta, 34518
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AZMJ.AZMJ_75_18

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  Abstract 


Background Otitis media with effusion (OME) is a common disease among preschool children. However, its pathophysiology is not well known, and gastroesophageal reflux diseases were proposed to play a role.
Aim of the work To estimate the prevalence of Helicobacter pylori in OME and to measure pepsin/pepsinogen levels in effusion fluid.
Patients and methods A total of 60 children, 1–6 years, who presented with OME were included in the study. Myringotomy was performed in all cases with effusion in the middle ear, and a tympanostomy tube was placed for the patient. Samples were collected, and levels of pepsinogen were measured, and H. pylori was detected.
Results H. pylori infection was detected in 26.7% of studied patients (16 patients). In addition, there was a statistically significant increase of pepsinogen in ear fluid aspirate when compared with serum values. There was a significant increase of adenoidectomy and/or tonsillectomy in positive when compared with negative H. pylori groups (100.0 vs. 75.0%, respectively), and there was a significant increase of pepsinogen in ear fluid aspirate in positive when compared with negative cases (272.75±26.42 vs. 253.59±23.49, respectively).
Conclusion H. pylori was isolated from middle ear fluid aspirate in 26.7% of preschool children with OME. Gastroesophageal reflux was proposed to transmit this bacterium from the stomach through passage in Eustachian tube.

Keywords: gastroesophageal reflux disease, Helicobacter pylori, otitis media with effusion, pepsinogen


How to cite this article:
Ismaeil WF, Aldesoky MM. Detection of pepsinogen and Helicobacter pylori in preschool age children with secretory otitis media. Al-Azhar Assiut Med J 2018;16:270-4

How to cite this URL:
Ismaeil WF, Aldesoky MM. Detection of pepsinogen and Helicobacter pylori in preschool age children with secretory otitis media. Al-Azhar Assiut Med J [serial online] 2018 [cited 2020 Jul 15];16:270-4. Available from: http://www.azmj.eg.net/text.asp?2018/16/3/270/255861




  Introduction Top


Otitis media with effusion (OME) is defined as the accumulation of serous or mucoid fluid inside the middle ear cleft, with no clinical manifestations of acute infection. It is a common disease in children, as it affects ∼20% of children after common cold infection and usually [if persisted for ≥3 months (long-term OM)] leads to a treatable childhood deafness all over the world [1],[2].

It has been reported that every child complains of at least one episode of OME by the age of 3 years [3],[4].

The etiology and pathogenesis of OME are not fully recognized, and it is thought to be multifactorial, with gastroesophageal reflux disease (GERD) as a possible etiology [5],[6],[7].

The causative role of GERD in pathogenesis of OME seems reasonable, as gastric reflux to the nasopharynx and subsequently to the middle ear is possible owing to Eustachian tube angulation and functional immaturity in children. Reflux leads to inflammatory reaction, which creates an ideal conditions for obstruction and bacterial accumulation [8].

Helicobacter pylori is a gram-negative, microaerophilic, spiral micro-organism that was discovered by Marshall et al. [9] from ulcers of the stomach.

Stomach is a specific location for H. pylori [10].

H. pylori has been isolated in patients with adenoids or tonsils, proposing that both adenoids and tonsils function as reservoirs of H. pylori [11],[12]. In addition, H. pylori have been detected in the mucosa of sinuses in chronic sinusitis [13],[14].

To confirm the relationship between GERD and OME, it is reasonable to search for pepsin/pepsinogen in middle ear fluid. Pepsin often exists in the form of deactivated form (pepsinogen), which is secreted mainly by mucosal cells of the stomach and is completely deactivated in alkaline environments such as the middle ear cavity. Normally, pepsin/pepsinogen is not detected in the middle ear. However, pepsinogen may be activated to pepsin under stimulation of hydrochloric acid in gastric juice during GERD [15].


  Aim of the work Top


The present work was designed to estimate the prevalence of H. pylori in OME and to measure pepsin/pepsinogen levels in effusion fluid.


  Patients and methods Top


The study included 60 children, aged 1–6 years, who presented with OME to ear, nose, and throat department of Al-Azhar Faculty of Medicine (New Damietta), during the period extending from March 2016 to March 2018. Diagnosis of OME was based on its clinical signs (a dull, vascularized, or retracted tympanic membrane at otoscope examination) or tympanogram type B up to 3 months after treatment.

Inclusion criteria

The present work included children aged 1–6 years, with the existence of middle ear fluid at least in one ear on clinical examination for at least 3 months.

Exclusion criteria

Children with cleft palate, cerebral palsy, Down syndrome, neurological delay, primary ciliary dyskinesia, and receiving antibiotics or gastrointestinal medications were excluded.

Ethical consideration

The study protocol was explained for parents or guardians of each child, and an informed consent was obtained. In addition, the study protocol was approved by the ethical and research committee of Al-Azhar University Hospital.

Detection of Helicobacter pylori in serous fluid

Myringotomy was performed in all cases with effusion in the middle ear, and a tympanostomy tube (VT) was placed for the patients. In addition, adenoidectomy or tonsillectomy was done at the same sitting if indicated. All surgical interventions were done under general anesthesia.

Two samples were collected and then delivered immediately to the microbiology unit. Each sample was placed into 0.5 ml of glycerol-supplemented brain–heart infusion broth, with each sample in individual microtube. One sample was examined by gram-stained film and then cultured on a blood agar base selective media as described by Melake et al. [7].

In addition, the second sample was stored at −20°C until extraction of DNA for H. pylori. Extraction was done by QIAamp DNA mini kit from Qiagen Company (Catalog no. 51304, Hilden, Germany) by Spin Protocol, as described by Farhadi and Tabatabaei [14].

Determination of pepsinogen

The presence of pepsinogen was determined by ELISA using the human pepsinogen I ELISA kit, as described by Buyruk et al. [16]. Venous blood sample was drawn at entry to surgical ward, and serum was separated and stored at −20°C till assay of pepsinogen was carried out. Values of effusion fluid and serum values were compared.

Statistical analysis

Software package (statistical package for social science, SPSS Inc., Chicago, Illinois, USA), version 20, was used for analysis. Quantitative data were presented as arithmetic mean±SD, whereas categorical data were expressed as frequency and percentage. Paired t test was used to compare between fluid and serum values of pepsinogen, whereas Student’s t and χ2 test were used to compare between positive and negative H. pylori subgroups for quantitative and qualitative variables. P value less than 0.05 was considered significant.


  Results Top


In the present work, age ranged from 1 to 6 years, and the mean age was 4.35±1.10 years. Overall, 55.0% of studied children were males, 61.7% were from urban city, most children had adenoid-tonsillectomy (38.3% previous and 45.0% current), and previous insertion of tympanostomy tube was reported in 18.6% of children ([Table 1]).
Table 1 General characteristics of studied children

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The examination of tympanic membrane revealed that it was vascularized in 43.3%, dull in 38.3%, and retracted in 18.3%, whereas the aspirated fluid nature was mucoid in 46.7%, and serous in 53.3%. H. pylori was detected in 26.7% of studied patients (16 patients). In addition, there was a statistically significant increase of pepsinogen in ear fluid aspirate when compared with serum values (258.70±25.54 vs. 99.23±6.06 µg/ml, respectively) ([Table 2]).
Table 2 Findings in the current intervention in studied children

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We had used PCR as gold standard for detection of H. pylori. A total of 16 patients were diagnosed by PCR; 12 (75%) of them were positive by direct film, with four (25%) false-negative result, with sensitivity, specificity, positive predictive value, and negative predictive value of 75, 100, 100, and 91.7%, respectively. In addition, 13 (81.25%) samples were detected by culture, with three (18.75%) false-negative cases, with specificity, sensitivity, positive predictive value, and negative predictive value of 81.25, 100, 100, and 93.6%, respectively, as shown in [Table 3]
Table 3 Results of diagnostic methods of Helicobacter pylori

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When comparing between positive and negative H. pylori subjects regarding studied parameters, we only found significant increase of adenoid-tonsillectomy in positive when compared with negative groups (100.0 vs. 75.0%, respectively). In addition, there was significant increase of pepsinogen values in ear fluid aspirate in positive when compared with negative cases (272.75±26.42 vs. 253.59±23.49, respectively). Otherwise, no other significant difference was found between positive and negative H. pylori groups ([Table 4]).
Table 4 Comparison between positive and negative Helicobacter pylori results regarding the studied parameters

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  Discussion Top


In the present work, the prevalence of H. pylori in OME was reported to be 26.7%, and this is lower than that reported by Saki and Rahim [12] who reported a prevalence rate of 42.6% when H. pylori was detected by PCR. The possible explanation for this high prevalence may be attributed to the fact that all their patients had adenoiditis or tonsillitis. In addition, the method of detection could be different between both studies, and they included children up to 10 years of age, whereas the upper limit of age in the present work is 6 years.

Yilmaz et al. [17] reported a prevalence rate of 67% (12 out of 18 patients), whereas Yilmaz et al. [18] in another study reported a prevalence rate of 47%. Agirdir et al. [19] used a urease test in 30 patients who complained of OME and adenoid hypertrophy and reported a positive rate of 66.6%. The possible explanation for these high rates could be owing to difference in test used for detection of H. pylori.

On the contrary, Boronat-Echeverria et al. [8] detected H. pylori in middle ear effusions in 5.7% with ELISA and 2.9% with PCR, and other publications reported that positivity varies from 14 to 18% [10],[20],[21],[22],[23], which are lower than reported in the present work. In addition, Karlidag et al. [24] reported a rate to be ranged from 16.3 to 35.5% when PCR was performed. Again, these wide variations may be owing to different methods used for diagnosis of H. pylori, racial differences, different sample sizes, or different age.

In our study, PCR was used as gold standard for diagnosis of H. pylori, as most of the previous studies, taking in consideration the importance of culture and direct film for low-income countries, limited equipment in laboratories, and isolated organisms could also be used for further research as culture, sensitivity, genotyping, and tracing the source of infection.

Anyway, and whatever the value of prevalence of H. pylori reported in different studies, these studies together confirmed the presence of H. pylori in OME, and the most accepted route for transmission is through the reflux. The next step in our study confirmed this assumption.In the present work, an interesting finding was significant higher concentration of pepsinogen of middle ear aspirate when compared with levels of serum samples. These results are comparable to those reported by Buyruk et al. [16]. The importance of these findings is that, it supports the transmission of gastric juice to middle ear and refutes the transfusion hypothesis and linked these levels with H. pylori infection, as positive group had significantly higher levels than negative group. Tasker et al. [25] included 65 patients and could identified pepsin/pepsinogen in 90.7%, with value extended between 0.8 and 213.9 µg/ml. However, they have a drawback of using porcine stomach-bound antibodies, which may react with several blood proteins and give false results. In addition, Lieu et al. [26] used ELISA and reported a pepsin/pepsinogen levels of 1.33 to 275 µg/ml in 70% of studied subjects. Crapko et al. [27] used the Western blot method (concentration range, 80–1000 ng/ml).

In summary, H. pylori was isolated from middle ear fluid aspirate in 26.7% of preschool children with OME. Gastroesophageal reflux was proposed to transmit this bacterium from the stomach through passage in the Eustachian tube. This is supported by high levels of pepsin/pepsinogen in the aspirate. Resistance to treatment may be owing to H. pylori, and future studies are needed to examine the role of H. pylori eradication on OME treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

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



 

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