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Saudi Pharmaceutical Journal : SPJ logoLink to Saudi Pharmaceutical Journal : SPJ
. 2012 Mar 3;20(4):345–353. doi: 10.1016/j.jsps.2012.02.004

Incidence of Helicobacter pylori infection and their clarithromycin-resistant strains in otitis media with effusion regarding phenotypic and genotypic studies

Nahla A Melake a,⁎, Ghada H Shaker a, Magdy A Salama b
PMCID: PMC3745192  PMID: 23960809

Abstract

Helicobacter pylori (H. pylori) are pathogenic bacteria that infect a half of the human population, colonize gastric mucosa and can be found in gastric juice. Reflux of gastric juice has been suggested to be associated with glue ear in children. It has been suggested that tonsil and adenoid tissues are potential reservoirs of H. pylori infection. These observations raise the question as to whether H. pylori infection might have a role in otitis media with effusion (OME) in children. The objectives of this research were to evaluate the incidence and possible role of H. pylori in the pathogenesis of OME in children and to evaluate the clarithromycin-resistant strains. Molecular assessment was done to evaluate the culture results vs. molecular study. A total of 60 children, who were prone to ventilation tube insertion, adenoidectomy and/or tonsillectomy were included in the study. The control group consisted of 40 children who underwent adenoidectomy and/or tonsillectomy without the history of OME. Samples of the middle ear fluid and mucosa, adenoid tissue, tonsillar tissue and gastric lavage were cultured and underwent polymerase chain reaction (PCR) analysis then were assembled by using QIAxcel System as capillary electrophoresis for H. pylori detection. There was significant difference between the results of cultures and PCR (P < 0.05). Middle ear fluid culture was positive for H. pylori in 40% of the patients vs. 56.7% PCR results while middle ear mucosa culture was positive in 20% vs. 26.7% PCR results. Gastric lavage culture was positive in 46.6% of the patients and PCR was positive in 63.3% of the patients. Adenoid culture and PCR were positive in 56.3% for each, while tonsil culture was positive in 70% and PCR was positive in 90%. H. pylori presence in the gastric lavage, the tonsillar and adenoid tissues by culture and PCR was significantly more frequent in the study group compared to the control group. The minimum inhibitory concentration (MIC) values of clarithromycin-resistant isolates ranged from 1.5 to 8 μg/ml. This study showed the presence of H. pylori in around 50% of the patients with OME. PCR revealed its sensitivity than culture techniques. The incidence of clarithromycin resistance was found to be high among the isolates (39.6%).

Keywords: Helicobacter pylori, Otitis media with effusion, Clarithromycin resistance, Polymerase chain reaction, Capillary electrophoresis

1. Introduction

The non-suppurative form of otitis media with the middle ear effusion is called otitis media with effusion (OME) or secretory otitis media (SOM). OME is one of the most frequent diseases of the ear in childhood (Gulani et al., 2010).

Helicobacter pylori infections are almost always acquired in childhood and if untreated, remain lifelong. In the developing world, up to 70% of children are infected with H. pylori by 15 years of age (De Giacomo et al., 2002). It is one of the most frequent infections all over the world. The prevalence of the disease is 30–40% in the developed countries and 80–90% in the developing countries. Crowded families, ethnical status, migration to the endemic places, the frequency in the family and general condition of the patient are the risk factors. It is not very clear how the disease infects somebody but oral–oral, fecal-oral or gastro-oral contamination may be possible. There are many clues of colonization in the dental plaques, tonsils and adenoid tissues (Nguyen et al., 2000).

Although OME is such a common disease of childhood, its pathogenesis still remains unsettled (Gulani et al., 2010). Causes of OME are believed to be multifactorial, including viruses, allergy, bacteria and their products, and dysfunction of the eustachian tube. Gastroesophageal reflux could also be a cause of this disease. Reflux of gastric contents from the nasopharynx to the middle ear is possible because of the angle between them and immaturity of the eustachian tube in children and infants and the supine position in which infants are often placed (Ruhani et al., 1996).

Tasker et al. (2002a) showed pepsinogen and pepsin in the middle ear fluid of patients with OME, indicating that gastric fluid could reach as far as the middle ear and this might be involved in the pathogenesis of OME. If gastric juice could enter the middle ear, H. pylori, a common inhabitant of gastric juice and mucosa in patients with peptic ulcer and chronic gastritis, would also be expected to be found in the middle ear of patients with OME.

H. pylori are microaerophilic, Gram-negative spiral organisms where many diagnostic assays have been developed; culture, histology, rapid urease test, urea breath test, serology, stool antigen test, and molecular-based tests (Vaira et al., 2002). Culture has the great advantage of permitting subsequent determination of the antimicrobial susceptibility of the strain isolated. However, disadvantages of culture include special conditions for specimen transportation, the use of complicated media with special conditions for maintenance, the need for special incubation conditions, and the length of time necessary to obtain a result (Tankovic et al., 2001).

Clarithromycin, a new generation of macrolide, has anti-H. pylori activity and may replace metronidazole in eradication regimens. Clarithromycin acts by binding to the peptidyl transferase region of 23S rRNA and inhibits bacterial protein synthesis (Houben et al., 1999). Preliminary studies on clinical trials with this drug alone or in combination with proton pump inhibitors and antibiotics achieved eradication rates varying from 40% to 100%. Resistance to clarithromycin has been observed and it may be an important factor in determining treatment failure, resulting in the varying eradication rates (Perez-Perez, 2000). The prevalence of H. pylori resistance to clarithromycin varies among different countries, such as 12% in Japan, 1.7–23.4% in Europe, and 10.6–25% in North America (Toracchio and Marzio, 2003; Mégraud, 2004). The difference depends on macrolide consumption. Several studies suggest that clarithromycin resistance is higher in strains obtained from children than in those from adults (Alarcón et al., 2003) because children have been more exposed to macrolides.

This study was undertaken to (i) evaluate the possible role of H. pylori in pathogenesis of OME in children; (ii) establish agar dilution method for the determination of H. pylori susceptibility to clarithromycin; (iii) assess the incidence of clarithromycin-resistant H. pylori among all yields isolated strains; and to (iv) conduct molecular assessment and compare its sensitivity vs. culture techniques.

2. Patients and methods

2.1. Patients

Permission of the patients’ parents and approval from the local Ethic Committee were obtained for the use of the specimens. The patients’ specimens were taken from ear–nose–throat (ENT) department in Riyadh National Hospital, Riyadh, Saudi Arabia. The study was performed between September 2010 and October 2011. The study group consisted of 60 children. They had undergone unilateral or bilateral ventilation tube insertion, adenoidectomy and/or tonsillectomy with a diagnosis of OME, adenoid hypertrophy and chronic tonsillitis. Before surgery, all patients underwent urea breath test (UBT) and serum samples were also tested for the presence of H. pylori IgG antibody by using quantitative enzyme immunoassay (EIA) (Monobind 1425-300 USA). The control group consisted of 40 children. They had undergone adenoidectomy and/or tonsillectomy without the history of OME. We used such a control group because it would be an ethical problem to use healthy subjects. Children with systemic diseases, those who received antibiotic or antacid treatment during the previous month and those who underwent any operation to the ear or pharynx were excluded. The diagnosis of OME was made by finding a dull, vascularized, or retracted tympanic membrane at ENT examination. The diagnosis was supported with type B tympanogram and audiogram that indicated conductive hearing loss. The lack of response to medical treatment with oral antibiotics and oral or topical decongestants for 3 months was an indication for ventilation tube insertion. OME was unilateral in 12 (20%) patients and bilateral in 48 (80%) patients. Adenoid hypertrophy and chronic tonsillitis were diagnosed by history, ENT examination and endoscopic examination. All operations were carried out with the patient under general anesthesia. After induction of general anesthesia, gastric lavage was performed for both groups. Orogastric nelaton catheter was inserted into the stomach and 10 cc of saline solution was given and subsequently collected back into the injector. A pH of gastric lavage fluid was measured with pH test strips (Sigma) and was immediately neutralized with 0.67 M Tris buffer (pH 7.4) and centrifuged at 13,000 rpm for 5 min at room temperature and each deposit was concentrated to a volume of 1 ml for further use. After cleaning the ear canal with an antiseptic solution and covering around the auricle with surgical drapes, a myringotomy was done with the use of a microscope. For the study group, after hemostasis, the middle ear fluid was aspirated and collected in the Juhn Tym-Taps (Xomad, Jackson-ville, FL, USA), which is a commercially available suction and collection device. The effusions were mucoid or seromucoid in character. After the effusion was aspirated, a small biopsy was taken from the promontorium mucosa with a cup forceps. For patients and control groups, 5 mm deep tissue specimens were obtained from the tonsil and adenoid tissues.

2.2. Isolation and identification of H. pylori from clinical samples

The specimens taken from patients (middle ear effusion, promontorium mucosa, adenoid, tonsil and gastric lavage) and from control group (adenoid, tonsil and gastric lavage) were transported immediately to the microbiology laboratory in 0.5 ml of glycerol-supplemented brain–heart infusion broth in individual microtubes. Biopsy materials were grounded by a sterile glass grinder. One half of the homogenate was used for culture and the other half was kept at −80 °C until completion of further molecular study. Cultures were performed on a commercialized Columbia Blood Agar Base (Oxoid) supplemented with 10% horse blood (bioMe´rieux) plus H. pylori selective supplement (Dent, SR0147E), under microaerophilic conditions at 37 °C in anaerobic jar flushed with a microaerophilic gas mixture (6% O2, 10% CO2, and 84% N2) for a maximum of 6 days. H. pylori isolates were defined as Gram-negative, spiral-shaped bacilli that were catalase and oxidase positive and rapidly (less than 1 h) urease positive.

2.3. Determination of susceptibility pattern of H. pylori to clarithromycin

MICs of clarithromycin (Hikma Pharmaceuticals, Saudi Arabia) were determined by using agar dilution method (Kim et al., 2004). Briefly, the bacteria were subcultured on Mueller–Hinton agar plates (Oxoid) supplemented with 10% horse blood (bioMe´rieux) for 48 h. The bacterial suspension, adjusted to 1 × 108 CFU, was inoculated directly onto each antibiotic-containing agar dilution plate according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2007). After 72 h of incubation at 37 °C under microaerophilic conditions, MIC was determined. The MIC was recorded as the lowest concentration of clarithromycin inhibiting the visible growth of H. pylori. Quality control was performed using H. pylori ATCC 43504. The resistance breakpoint for clarithromycin was set at MIC ⩾ 1.0 μg/ml, intermediate if MIC = 0.5 μg/ml and sensitive at MIC ⩽ 0.25 μg/ml (CLSI, 2007).

2.4. DNA extraction and PCR amplification

DNA extraction from both cultured isolates and specimens was performed using the illustra™ bacteria genomic Prep Mini Spin Kit (Amersham Place, Little Chalfont, Buckinghamshire, UK). Amplification was done by using forward primer 5′-GCCAATGGTAAATTAGTT-3′ and reverse primer 5′-CTCCTTAATTGTTTTTAC-3′ to amplify a 411-bp internal fragment of the urease A gene of H. pylori. This assay had been assessed previously for its specificity for the urease A gene of H. pylori and was not found to cross-react with other Helicobacter sp. or known other urease-producing organisms (Allaker et al., 2002). PCR conditions were: one cycle at 98 °C for 3 min followed by 30 cycles at 98 °C for 1 min, 54 °C for 1 min, and 74 °C for 1 min. A final elongation step was at 74 °C for 15 min, using Mycycler TM Thermal cycler (BioRad, USA). A DNA ladder (15.0–1000.0 bp) was used to estimate allele sizes in base pairs (bp) for the gel. The amplified PCR products were transferred directly from the thermocycler into the sample tray of the QIAxcel System (QIAGEN®, Germany). Separation was performed using the AM320 method (100 ng/μl sample was injected at voltage 5 kV for 10 s, and separation voltage was 6 kV for 320 s). Under these conditions, the negatively charged nucleic acid molecules migrated through the capillary to the positively charged end. As the molecules migrated through the capillary, they passed a detector which detected and measured the fluorescent signal. A photomultiplier detector converted the emission signal into electronic data, which were transferred to the computer for further processing using QIAxcel ScreenGel Software. After processing, the data were presented as both simulated bands on gel images and peaks in electropherograms. The reference strain H. pylori ATCC 43504 was used as control.

2.5. Statistical analyses

Computer SPSS program version 15 was used. The results were expressed by applying Chi-square test and P values. P value < 0.05 was considered to be significant.

3. Results

The study group consisted of 60 children who were between ages 2 and 14 years (median 8.3). Thirty-four (56.7%) were male and 26 (43.3%) were female. The control group consisted of 40 children who were between ages 4 and 16 years (median 8.9) (50% male and 50% female). The mean ages and sex rates of both groups were not significantly different (P > 0.05).

3.1. Demographic features and clinical diagnosis of patients and control cases

Table 1 summarizes the demographic features of patients who had undergone myringotomy and control cases which included; age, gender, residence (place of stay), income of the family, and father’s education levels. No relationship was found between these variables and colonization of H. pylori in the middle ear in children (P > 0.05). Bilateral myringotomy was more indicated than unilateral surgery (P < 0.05) and was associated with adenotonsillectomy in 53.3% of the patients.

Table 1.

Demographic features in patient’s who underwent myringotomy (No. = 60) and control cases (No. = 40).

Variables Group Number (%) P value
Age Patients Preschool age (<6 years) 24 (40) P > 0.05
School age (⩾6 years) 36 (60)
Control Preschool age (<6 years) 18 (45)
School age (⩾6 years) 22 (55)
Gender Patients Male 34 (56.7) P > 0.05
Female 26 (43.3)
Control Male 20 (50)
Female 20 (50)
Residence Patients City (Urban) 33 (55) P > 0.05
Village (Rural) 27 (45)
Control City (Urban) 25 (62.5)
Village (Rural) 15 (37.5)
Father’s education Patients Uneducated 16 (26.7) P > 0.05
Primary school 7 (11.7)
High school 18 (30)
Diploma 11 (18.3)
Two years after diploma 2 (3.3)
Bachelor (of degree) 5 (8.3)
Ph.D. 1 (1.7)
Control Uneducated 9 (22.5)
Primary school 7 (17.5)
High school 10 (25)
Diploma 9 (22.5)
Two years after diploma 4 (10)
Bachelor (of degree) 1 (2.5)
Ph.D. –
Myringotomy site Patients Unilateral 12 (20) P < 0.05
Bilateral 48 (80)
Adenotonsillectomy Patients Previously done 10 (16.7) P < 0.05
Associated with myringotomy 32 (53.3)
Not indicated 18 (30)
Control Indicated 40 (100) –
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3.2. Identification results of H. pylori among patients and control groups

Tables 2 and 3 show the results of indirect diagnostic tests of H. pylori (UBT and IgG test), culture and PCR analysis among different specimens of the studied group and control group, respectively. UBT was positive in 38 patients (63.3%) and the serology test for IgG antibody was positive in 36 (60%) of the patients. The indirect diagnostic tests of H. pylori showed significant difference among studied cases (P > 0.001). Middle ear fluid culture was positive for H. pylori in 24 (40%) patients and PCR was positive in 34 (56.7%) patients, middle ear mucosa culture was positive in 12 (20%) patients and PCR was positive in 16 (26.7%) patients, and gastric lavage was positive in 28 (46.6%) patients and PCR was positive in 38 (63.3%) patients. Adenoid culture and PCR were positive in 18 out of 32 patients (56.3%) for each. While tonsil culture was positive in 14 out of 20 patients (70%) and PCR was positive in 18 out of 20 (90%) as shown in Table 2. There was significant difference between the results of cultures and PCR (P > 0.05). Referring to control group, UBT was positive in 8 cases (20%), and the serology test for IgG antibody was positive in 8 (20%) of the cases. The indirect diagnostic tests of H. pylori showed no significant difference among control cases (P > 0.05). Gastric lavage culture was positive in 6 (15%) patients and PCR was positive in 8 (20%) patients. Adenoid culture was positive in 4 (10%) patients and PCR was positive in 6 (15%) patients. While tonsil culture was positive in 10 (25%) patients and PCR was positive for 12 (30%) patients as shown in Table 3. H. pylori presence in the gastric lavage, the tonsillar and adenoid tissues by culture and PCR was significantly more frequent in the study group compared to the control group (P = 0.007). All PCR positive specimens for gastric H. pylori patients showed positive UBT (63.3%). While, 60% of our patients showed PCR positive specimens for gastric H. pylori and positive IgG antibody test. Also, all PCR positive specimens for gastric H. pylori of control cases showed positive UBT (20%) and positive IgG test antibody (20%). Fig. 1a and b shows the capillary electrophoresis results of one isolate (No. 26) as an example by using QIAxcel System and QIAxcel ScreenGel Software, as both simulated bands on gel images and peaks in electropherograms. Gel images of amplified PCR products gave one DNA band at 411-bp for all positive specimens (positive middle ear fluid, gastric lavage, adenoid tissue and tonsilar tissue) (Fig. 1a). Electropherogram showed that all peaks were overlapping at PCR products sized 411-bp (Fig. 1b).

Table 2.

Identification results of H. pylori among the study group.

Patient number Indirect diagnostic tests
 Culture and molecular identification of specimens
Middle ear fluid
 Middle ear mucosa
 Gastric lavage
 Adenoid
 Tonsil
UBT Serology test (IgG) Culture PCR Culture PCR Culture PCR Culture PCR Culture PCR
1,5,16,20, 31,36,54,57 + + + + − − + + − − N N
2,17,43,60 + + + + + + + + N N N N
4,19,32,45 + + + + − − + + + + + +
6,21,47,59 + + + + + + + + + + − +
7,10,40,42 + + − − − − − + N N N N
8,23,33,56 − − − − − − − − − − N N
9,24,38,52 + + + + + + + + + + + +
11,51 − − − − − − − − − − − −
12,22,35,46 + + − + − − + + N N N N
3,13,14,15,18,25,28,29,34,39,41,44,49,53,55,58 − − − − − − − − N N N N
26,37 + − − + − − − + + + + +
27,30,48,50 + + − + − + − + + + + +
Ratio (%) 38/60 (63.3) 36/60 (60) 24/60 (40) 34/60 (56.7) 12/60 (20) 16/60 (26.7) 28/60 (46.6) 38/60 (63.3) 18/32 (56.3) 18/32 (56.3) 14/20 (70) 18/20 (90)
P-value P > 0.001 P > 0.05
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+, positive H. pylori culture, PCR, UBT and IgG.

−, negative H. pylori culture, PCR, UBT and IgG.

N, no specimen.

Table 3.

Identification results of H. pylori among the control group.

Patient number Indirect diagnostic tests
 Culture and molecular identification of specimens
Gastric lavage
 Adenoid
 Tonsil
UBT Serology test (IgG) Culture PCR Culture PCR Culture PCR
1,36 + + + + + + + +
2,5,6,8,9,10,12,15,16,18,19,20,22,23,24,26,27,29,30,32,33,34,35,38 − − − − − − − −
3,11,13,21,31,37 − − − − − − + +
4,14,25,39 + + + + − − − −
7,40 − − − − + + − +
17,28 + + − + − + + +
Ratio (%) 8/40 (20) 8/40 (20) 6/40 (15) 8/40 (20) 4/40 (10) 6/40 (15) 10/40 (25) 12/40 (30)
P-value P > 0.05 P > 0.001
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+, positive H. pylori culture, PCR, UBT and IgG.

−, negative H. pylori culture, PCR, UBT and IgG.

Figure 1.

Figure 1

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Electropherogram and gel images of amplified PCR products sized 411-bp by using QIAxcel System and QIAxcel ScreenGel Software of studied isolate No. 26. (a) Gel image reveals that; lane 1, H. pylori ATCC 43504 (positive control strain); lane 2, positive middle ear fluid; lane 3, negative middle ear mucosa; lanes 4, 5, and 6, positive gastric lavage, adenoid tissue and tonsilar tissue, respectively. (b) Electropherogram is a plot of the time (min) from injection against the detector signal (fluorescence intensity). First and last peaks in electropherogram indicate internal alignment markers (15.0–1000.0 bp). Each peak between the alignment markers indicates the presence of one strain genotype. All the peaks are overlapping at PCR products sized 411-bp.

3.3. Clarithromycin resistance

A total of 96 H. pylori were isolated from all patient samples (232 specimens) (41.4%). Thirty-eight of these isolates (39.6%) were resistant to clarithromycin (MIC ⩾ 1.0 μg/ml), 4 isolates (4.2%) showed intermediate resistance (MIC = 0.5 μg/ml) and 54 isolates (56.2%) were sensitive (MIC ⩽ 0.5 μg/ml) to clarithromycin. Twenty H. pylori strains were isolated from control specimens (120 specimens) (16.7%). Four of these isolates (20%) were resistant to clarithromycin and 16 isolates (80%) were sensitive (Fig. 2). The MIC values of all the clarithromycin-resistant isolates ranged from 1.5 to 8 μg/ml. There was a significant statistical difference between the patients and control isolates susceptible to clarythromycin (P > 0.001).

Figure 2.

Figure 2

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Clarithromycin susceptibility pattern among patients and control.

4. Discussion

H. pylori infection is usually acquired in childhood and may persist throughout the life of the patient. In this study, 63.3% of the children were infected before/at 14 years of age revealing high rate of H. pylori infection in the first and second decades of life. Indirect diagnostic tests such as UBT and serology tests were positive in 63.3% and 60% of the patients, respectively. This had to be concomitant with the PCR results obtained in this study and the results of Lascols et al. (2003) and Zahedi et al. (2009). Bilateral myringotomy was indicated in 80% of the patients and associated with adenotonsillectomy in 53.3% revealing the same reason of compliant in both infections (adenotonsillitis and OME).

Regarding the PCR results, we reported that H. pylori were present in 56.7% of the middle ear effusion, 90% of the tonsillar tissue and 70% of the adenoid tissue of the patients. This was in agreement with the results of Unver et al. (2001), Agirdir et al. (2006) and Fancy et al. (2009), they suggested that H. pylori may be responsible for the etiopathogenesis of chronic OME because they had found H. pylori positivity in 57.89%, 66.6% and 32% of their patients who underwent adenotonsillectomy, by using campylobacter-like organisms (CLO) test and PCR analysis. Yilmaz et al. (2005) had found that, 67% of the chronic OME patients showed H. pylori presence in the middle ear effusions. But, they did not find any H. pylori presence in the adenoid tissues of the same patient group. Also, Yilmaz et al. (2006) reported significantly increased colonization by H. pylori of the middle ear, and tonsil and adenoid tissue in patients with OME which indicated that the bacteria might be involved in the pathogenesis of OME. Morinaka et al. (2005) study supported our data as H. pylori strains were detected in 12 out of 15 smears of the middle ear effusion (80%) by immunohistochemistry study.

Our data demonstrated that, the study group had significantly more positive gastric fluid (63.3% vs. 20%), tonsilar tissue (90% vs. 30%) and adenoid tissue (56.3% vs. 15%) for H. pylori than the control group. These results indicated that, H. pylori were strongly associated with OME. In contrary, Yilmaz et al. (2004) reported that H. pylori were not detected in the tonsil and adenoid tissues of the 50 children with chronic or recurrent tonsillitis and adenoiditis. Additionally, Di Bonaventura et al. (2001) could not detect H. pylori positivity by the PCR method in the tonsil swabs and biopsy materials of 58% of the patients in which H. pylori was detected in gastric biopsy cultures. Skinner et al. (2001), could not detect H. pylori positivity in the tonsillectomy specimens of 50 patients of whom 14 (28%) were positive for H. pylori IgG by means of immunohistochemistry and CLO test. Sudhoff et al. (2008) and Ozcan et al. (2009) reported that, there was poor evidence for the existence of H. pylori-associated OME.

There were multiple studies supporting or opposing the role of H. pylori infections in OME, may be due to the particular characteristics of the population in which the studies were conducted, the diversity of the methods used or the sensitivity of H. pylori to cultural environmental conditions. Future studies with large numbers of patients are needed to elucidate the potential pathogenetic relationship between H. pylori and OME.

The sensitivity of H. pylori culture of the collected specimens was lower than that of PCR analysis, UBT and IgG antibody tests. This was supported by Pitkaranta et al. (2005), who revealed that PCR is a very convincing method to demonstrate the existence of live bacteria. So, culture is not a gold standard technique for the determination of H. pylori. PCR analysis and UBT are the most sensitive techniques followed by IgG antibody test.

The demonstration of H. pylori in the middle ear, adenoid, and tonsillar tissue was most probably related to reflux of gastric contents to pharynx. Acid reflux leads to mucosal inflammation and edema in the nasopharynx, disturbing eustachian tube clearance, thus causing the nasopharyngeal bacteria such as H. pylori to enter the middle ear. This was supported by higher adenoid and tonsillar tissue colonization by H. pylori in this study. Adenoid and tonsillar tissue act as reservoirs of infection disseminating H. pylori to the middle ear through the eustachian tube in patients with OME. This was supported by significantly increased H. pylori colonization in the adenoids and tonsils of patients with OME compared with those with adenotonsillar hypertrophy without OME (56.3% vs. 10% for adenoid and 70% vs. 25% for tonsils, respectively). The middle ear is another site that H. pylori can live in. There is a microaerophilic environment in the middle ears of OME patients which is very appropriate for H. pylori to reproduce. It has been shown that the pH value of the middle ear effusions of the chronic OME patients is between 7 and 9. This is in agreement with Steven (2005), but still the exact mechanism remains unclear.

The data were supported by the studies of the others Rozmanic et al. (2002), who recorded the incidence of gastroesophageal reflux disease as 55.6% in the patients of chronic tubotympanic disease. Also, Velepic et al. (2000) noted that, gastroesophageal reflux disease was presented in 60% of the chronic tubotympanic patients and Tasker et al. (2002b) showed that gastric fluid reflux was presented in 83% of the middle ear effusions of glue ears. Carr et al. (2001) demonstrated that, 42% of 95 children younger than 2 years who underwent adenoidectomy had gastroesophageal reflux disease. In an animal study, White et al. (2002) had demonstrated that, nasopharyngeal exposure to HCl and pepsin in concentrations similar to that found in gastric reflux leads to the impairment of ventilatory function and mucociliary clearance of the eustachian tube. Gastroesophageal reflux disease has been considered a contributing factor for many nasopharyngeal disease processes occurring anterior to the eustachian tube (Phipps et al., 2000). Studies by Poelmans et al. (2002) and Tasker et al. (2002b) showed that, gastroesophageal reflux disease was a major pathology in intractable chronic OME and antireflux treatment was effective in dissolving the problem.

Gastric lavage is a less invasive but less sensitive method to take gastric fluid. However, because we used the same method for both study and control groups, the comparisons would be valid. Furthermore, the validity of gastric lavage in detecting H. pylori in the stomach was supported by finding significantly increased colonization of the middle ear, adenoid and tonsillar tissue with H. pylori parallel to the gastric lavage fluid. All our patients whose middle ear was positive for H. pylori also had the bacteria in their gastric fluid. This finding indicated that H. pylori might have reached the middle ear.

Clarithromycin is the most effective drug used in the eradication of infection by H. pylori. Due to worldwide increase in resistance, pre-treatment susceptibility testing for clarithromycin is recommended. The incidence of clarithromycin resistance was found to be high among the isolates in the present study (39.6%). This result was in agreement with Samra et al. (2002), who revealed that clarithromycin resistance was 38.2% in H. pylori isolates from his patients. Similar results but with less resistance extension pattern was reported by Lee et al. (2005), who detected that H. pylori is resistant to clarithromycin in 20.2% of their isolates. Moreover, H. pylori strains that were resistant to clarithromycin have been reported increasingly in several studies and it was the most important cause of treatment failure (Torres et al., 2001; Toracchio and Marzio, 2003). Cultural environmental conditions may influence the results of susceptibility testing.

Owing to the small size of the group in this study, we propose that further studies be carried out with larger sample size in an effort to explain the presence of H. pylori in middle ear effusions and discuss the mechanisms of clarithromycin resistance.

Acknowledgment

This research project was supported by a grant from the research center of the center for female scientific and medical colleges in King Saud University.

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