Investigation of the association between glaucoma and Helicobacter pylori infection using the 14 C-urea breath test

Glaucoma is a neurodegenerative disease characterized by retinal ganglion cell degeneration. Although glaucoma is prevalent worldwide, its etiopathogenesis remains incompletely understood. Increased intraocular pressure (IOP) is the most frequently implied cause for retinal ganglion cell degeneration. Recent studies show that cytokines, impaired ocular blood flow, and structural changes in blood vessels are also involved in the pathogenesis of glaucomatous optic neuropathy. Helicobacter pylori is a helical, Gram-negative bacterium known to be involved in the pathogenesis of gastritis, gastric ulcer, gastric carcinoma, and Alzheimer’s disease. Some studies have also suggested that H. pylori infection may be involved in nongastrointestinal disorders because of the release of reactive oxygen species. Thus, it is thought that H. pylori facilitates glaucoma pathogenesis by causing the release of Nitric Oxide, endothelin-1, and reactive oxygen species. Several recent studies have investigated the association between H. pylori infection and glaucoma. In most such studies, serum IgG levels have been used to establish the presence of H. pylori infection. Recently, noninvasive and invasive methods have been used to evaluate the presence of H. pylori. Invasive methods include histopathological examination gastric mucosal samples, polymerase chain reaction, culture methods, and the rapid urease test. Noninvasive methods include serological tests, C-urea breath test (14C-UBT), and H. pylori antigen level measurements in stools. Accurate diagnostic rates of 80%-84% have been reported for noninvasive tests by the serological method; however, tests can be positive for 6-12 months after H. pylori eradication; thus, positivity does not necessarily confirm active infection. Because samples need to be stored at -20°C, the use of the H. pylori stool antigen method is restricted. The other noninvasive test is the 14C-UBT, which is the most common test reported in the literature. Many studies have reported a sensitivity of 95%-100% and a specificity of 97%-100% for 14C-UBT. Thus, in recent years, it has become the preferred method for detecting H. pylori infections. To the best of our knowledge, this is the first study to investigate the relationship between H. pylori and glaucoma using 14C-UBT.


INTRODUCTION
Glaucoma is a neurodegenerative disease characterized by retinal ganglion cell degeneration.Although glaucoma is prevalent worldwide, its etiopathogenesis remains incompletely understood.Increased intraocular pressure (IOP) is the most frequently implied cause for retinal ganglion cell degeneration (1) .Recent studies show that cytokines, impaired ocular blood flow, and structural changes in blood vessels are also involved in the pathogenesis of glaucomatous optic neuropathy (2) .
Helicobacter pylori is a helical, Gram-negative bacterium known to be involved in the pathogenesis of gastritis, gastric ulcer, gastric carcinoma, and Alzheimer's disease (3)(4)(5) .Some studies have also suggested that H. pylori infection may be involved in nongastrointestinal disorders because of the release of reactive oxygen species (6) .Thus, it is thought that H. pylori facilitates glaucoma pathogenesis by causing the release of Nitric Oxide, endothelin-1, and reactive oxygen species (7) .Several recent studies have investigated the association between H. pylori infection and glaucoma (8)(9)(10)(11) .In most such studies, serum IgG levels have been used to establish the presence of H. pylori infection.
Recently, noninvasive and invasive methods have been used to evaluate the presence of H. pylori.Invasive methods include histopa-thological examination gastric mucosal samples, polymerase chain reaction, culture methods, and the rapid urease test.Noninvasive methods include serological tests, 14 C-urea breath test (14C-UBT), and H. pylori antigen level measurements in stools (12) .Accurate diagnostic rates of 80%-84% have been reported for noninvasive tests by the serological method; however, tests can be positive for 6-12 months after H. pylori eradication; thus, positivity does not necessarily confirm active infection.Because samples need to be stored at -20°C, the use of the H. pylori stool antigen method is restricted.The other noninvasive test is the 14C-UBT, which is the most common test reported in the literature.Many studies have reported a sensitivity of 95%-100% and a specificity of 97%-100% for 14C-UBT (13,14) .Thus, in recent years, it has become the preferred method for detecting H. pylori infections (15,16) .To the best of our knowledge, this is the first study to investigate the relationship between H. pylori and glaucoma using 14C-UBT.

METHODS
The Ethical Committee Gaziantep University Hospitals, Gaziantep, Turkey provided the ethical approval for this study.The study group included 35 patients (mean age: 59.08 ± 9.82 years; range: 28-80 years; male/female: 20/15) diagnosed with primary open-angle glaucoma Investigation of the association between glaucoma and Helicobacter pylori infection using the 14 C-urea breath test RESUMO Objetivo: Investigar a associação entre glaucoma e infecção por H. pylori por meio do teste para a presença de infecção por H. pylori em pacientes com glaucoma usando o teste de respiração da ureia 14
Patients brushed their teeth before the test to prevent contamination from urease-producing resident flora in the mouth.After over night fasting (≥4 h), the patients swallowed 37 kBq (1 μCi) of an encapsulated form of 14 C (Helicap TM , Kibion, Uppsala, Sweden) with 50 mL of water.The breath samples were collected using a dry cartridge system (Heliprobe BreathCard TM , Kibion) 10 min afterward.The patients exhaled into the cartridge until the indicator membrane changed color from orange to yellow.The breath card was then inserted into a Geiger-Müller counter (Heliprobe TM analyser, Kibion) and the activity counted for 250 s.The results were expressed both as counts per minute (CPM) and as a grade (0: not infected, CPM <25; 1: equivocal, CPM: 25-50; 2: infected, CPM >50).
All analyses were performed using SPSS 15.0 software (IBM Corporation, Armonk, NY, USA).14C-UBT positivity was compared using Pearson's chi-square test.Continuous variables between groups were compared by t tests for normally distributed values (age).p values of <0.05 were considered significant.

RESULTS
Table 1 summarizes the demographic characteristics of both groups.The mean age in the glaucoma and control groups were 59.08 ± 9.82 and 58.77 ± 9.44 years, respectively.The mean age and gender were similar between the two groups.In addition, the incidence of dyspeptic symptoms was similar between the two groups.No adverse effects related to the 14C-UBT were observed.
Table 1 also presents the H. pylori and 14C-UBT statuses.No significant difference regarding the rates of H. pylori infection and 14C-UBT positivity were observed between the study and control groups (p=0.473).H. pylori was detected in 18 (52.9%)patients with glaucoma compared with 15 (47.1%) controls (Table 1).

DISCUSSION
Glaucoma is a multifactorial, progressive ocular condition with an unknown etiology.Previous studies have demonstrated that many risk factors could be involved in glaucoma pathogenesis.However, increased IOP is known to be a major risk factor (1) .H. pylori infection is also associated with gastrointestinal and other conditions (17) .In recent years, several studies have investigated the relationship between H. pylori infection and the pathogenesis of some ocular disorders, such as central serous chorioretinopathy (18,19) , glaucoma (8,9,10,11,20) , blepharitis, and uveitis.However, the present study failed to find an association between H. pylori infection and glaucoma.In the previous studies, a serological method (ELISA) was used to detect H. pylori infection; although ELISA is a simple and practical test, it shows high false-positive rates in the elderly and in those not having received H. pylori eradication treatment (21,22) .In addition, because elderly individuals comprise the majority glaucoma cases, ELISA was considered inappropriate.Furthermore, in a recent study, the sensitivity and spe cificity of the urea breath test were 91.4% and 93.8%, respectively, which was comparable with the values for histopathological examination in the elderly (23) .Therefore, we preferred the 14C-UBT method, which demonstrates high sensitivity (92%-93%), specificity (88%-93%), and accuracy (90%-93%), in addition to being noninvasive and reliable in the elderly (15,16) .
Deshpande et al. examined H. pylori antibodies in the aqueous humor and serum of patients with POAG, those with pseudoexfoliation glaucoma (PXFG), and controls (11) .Serum IgG positivity was significantly higher in the POAG group than in the control and PXFG groups; however, no significant differences in the antibody levels of the aqueous humor were found between the POAG and control groups.However, significantly higher antibody levels were found in the aqueous humor of the PXFG group than in the other groups (11) .Deshpande et al. 's study was limited by the older age of patients in the glaucoma group than in the control group.The higher positivity rate in glaucoma patients was expected because the serological method used has a high false-positive rate.In another study using the same method, Galloway et al. found no relationship between H. pylori infection positivity and glaucoma (24) .Similarly, no relationship was found between glaucoma and H. pylori infection positivity in a study by Kurtz et al., in which serology and cytotoxin-associated geneA (CagA) seropositivity were evaluated (25) .Handa et al. reported that the CagA gene may be associated with apoptosis, irregular gastric cell activity, and carcinogenesis (26) .In addition, H. pylori strains harboring CagA were reported to produce more interleukin-8 than those without CagA, causing higher rates of gastric inflammation and atrophy, by Israel et al. (27) .Kurtz et al. suggested that H. pylori has no effect on apoptosis that plays a role in the pathogenesis of glaucoma, because the CagA levels in patients with glaucoma were similar to those in the control group.However, Kountouras et al. advocated that H. pylori infection induces apoptosis and causes the development and progression of glaucomatous optic neuropathy by releasing several vasoactive and pro-inflammatory substances (28) .
In Turkey, the rates of serological H. pylori positivity in the general population are high, with rates of up to 68% in symptomatic cases (29) .In the present study, the rates of H. pylori positivity detected using the 14C-UBT method were in agreement with the previously reported levels.The finding of high rates of H. pylori positivity is expected in countries with higher rates of H. pylori positivity, such as Turkey.

CONCLUSION
Our study has some limitations.First, we did not compare our method with other H. pylori evaluation methods, such as the serological and stool H. pylori antigen methods, in patients with glaucoma and healthy subjects.Although the reliability of the tests could be compared when used in combination, the use of additional tests would have increased the cost.Second, our study included a limited number of subjects.Further comprehensive studies are needed to verify these results and investigate the association between glaucoma and H. pylori using multiple methods.