- Email: [email protected]
Relationship between Helicobacter pylori infection and glaucoma1
Relationship between Helicobacter Pylori Infection and Glaucoma Jannis Kountouras, MD, PhD,1 Nikolaos Mylopoulos, MD, PhD,2 Panagiota Boura, MD, PhD,1 Christos Bessas, MD, PhD,3 Dimitrios Chatzopoulos, MD,1 John Venizelos, MD, PhD,4 Christos Zavos, MD1 Objective: To determine the frequency of Helicobacter pylori (H. pylori) infection in glaucoma patients and in anemic control participants. Design: Prospective, nonrandomized, comparative study. Participants: The authors investigated 32 patients with chronic open-angle glaucoma (COAG), 9 patients with pseudoexfoliation glaucoma (PEG), and 30 age-matched anemic control participants. Methods: Upper gastrointestinal endoscopy was performed to evaluate macroscopic abnormalities, and gastric mucosal biopsy specimens were obtained for the presence of H. pylori infection tested by rapid urease slide test (CLO test) and by Cresyl fast violet staining, Giemsa staining, or both. The presence of gastritis was classified in accordance with the Sydney system by using hematoxylin and eosin stain. In addition, intestinal metaplasia was evaluated with Alcian blue stain. Saliva samples were also tested by CLO. Serum was analyzed for the presence of H. pylori-specific IgG antibodies by enzyme-linked immunosorbent assay. Main Outcome Measure: Histologic examination for the presence of H. pylori. Results: In 87.5% of the COAG patients, 88.9% of the PEG patients, and 46.7% of the anemic control participants, H. pylori infection was histologically confirmed (odds ratio, 8.00; chi-square, 11.81; P ⫽ 0.0006 and 9.14; chi-square, 5.01; P ⫽ 0.02, respectively). H. pylori was detected by urease test: (1) in the gastric mucosa in 71.9% of the COAG patients, in 77.8% of the PEG patients, and in 46.7% of the anemic control participants (P ⫽ 0.03 and P ⬎ 0.05, respectively); and (2) in the saliva in 37.5% of the COAG patients, in 55.6% of the PEG patients, and in 30% of the anemic control participants (P ⬎ 0.05). Sixty-eight percent of glaucoma patients and 30% of anemic control participants were seropositive for H. pylori (P ⫽ 0.002). When compared with anemic control participants, glaucoma patients exhibited less often endoscopic normal appearance of gastric mucosa (P ⫽ 0.01), and more often antral gastritis (P ⫽ 0.0004) or peptic ulcer disease (P ⫽ 0.01). Histologic grade 3 gastritis was observed only in the glaucoma patients (P ⫽ 0.03). Conclusions: H. pylori infection seems more frequent in glaucoma patients. If confirmed, this may indicate either a common factor that causes susceptibilities to both glaucoma and H. pylori infection or that H. pylori may be a causal factor for developing glaucoma. Ophthalmology 2001;108:599 – 604 © 2001 by the American Academy of Ophthalmology. Glaucoma is a progressive optic neuropathy that may have a multifactorial cause, which remains largely unknown.1–3 The most important risk factor is increased intraocular pressure. However, other factors may also damage the neural tissue in glaucomatous optic neuropathy. The most prominent of these is vascular dysregulation.2 According to recent evidence, this disease may be associated with changes in Originally received: January 31, 2000. Accepted: November 10, 2000. Manuscript no. 200038. 1 Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece. 2 Department of Ophthalmology, Ippokration Hospital, Thessaloniki, Greece. 3 Private practice, Thessaloniki, Greece. 4 Department of Pathology, Ippokration Hospital, Thessaloniki, Greece. Presented in part at the sixth congress of the European Glaucoma Society, London, England, June 2000. The authors have no commercial interests in the products or devices mention herein. Address reprints requests to Jannis Kountouras, MD, Gastroenterologist, 8 Phanariou Street, Byzantio, 551 33, Thessaloniki, Macedonia, Greece. E-mail: [email protected], [email protected]. © 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
endothelium-dependent vascular regulation.2,4 –10 In addition, impaired ocular blood flow resulting from blood hyperviscosity (increased platelet aggregation on atheromatous plaques in the elderly)11–18 and cytokines may also be involved in the pathogenesis of this disease.19 –21 A theoretical relationship between glaucoma and H. pylori infection exists: (1) both are diseases of older adults in the developed world1,22,23; (2) chronic H. pylori infection may produce systemic disorders involving vascular tone resulting from the release of vasoactive and proinflammatory substances24 –33; and (3) H. pylori infection is associated with arteriosclerosis-induced increased platelet activation and aggregation.34,35 The aim of the study was to investigate whether H. pylori infection is associated with glaucoma.
Patients and Methods Participants Group 1, the control participants, consisted of 30 asymptomatic patients who were undergoing upper gastrointestinal endoscopy ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00598-4
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Ophthalmology Volume 108, Number 3, March 2001 for the investigation of mild anemia but in whom endoscopy was normal. Group 2, patients with documented glaucoma, consisted of 32 patients with chronic open-angle glaucoma (COAG) and 9 patients with pseudoexfoliation glaucoma (PEG). The glaucoma patients were included in the study using the following criteria: a history of intraocular pressure of 21 mmHg or more; typical optic nerve head changes including saucerization, rim thinning or notching in the inferior or superior temporal area of the optic nerve head, or total glaucomatous cupping36; and typical visual field loss including a paracentral, arcuate, or Seidel’s scotoma or a nasal step.36 Seven of our glaucoma patients were legally blind in one eye as a result of glaucoma. Exclusion criteria included all eye diseases other than glaucoma, diabetes mellitus, and a myopic refractive error exceeding ⫺8 diopters. All 71 participants were older than 45 years and younger than 70 years. The intraocular pressures were measured with a calibrated Goldmann applanation tonometer. All the glaucoma patients had prior experience with automated perimetry. The visual fields were assessed with the Octopus program G1 (Octopus 500EZ G1 programme, Luterzeag AG, Zurich, Switzerland) by the same perimetrists. None of the glaucoma patients received oral medications that could decrease intraocular pressure (e.g., carbonic anhydrase inhibitors). The control participants had recently been diagnosed with mild iron deficiency anemia. The diagnosis of anemia was based on history and the gastrointestinal investigation, and none of the participants had received any treatment (e.g., ferrous sulfate) before the diagnosis. The blood profile of the anemic control group was: mean hematocrit 34.4 ⫾ 1.3%, mean hemoglobin 10.3 ⫾ 0.5 g/100 ml, and mean serum ferritin levels 9.5 ⫾ 1.7 g/l (mean ⫾ standard deviation). The causes of iron deficiency anemia were: hemorrhoids in nine participants, diverticulosis in five participants, blood donation in four participants, genitalia bleeding in four female participants, and inadequate iron supply in two participants. The cause of the anemia could not be determined in six participants. All 71 patients (41 glaucoma patients, 30 anemic control participants) underwent elective diagnostic upper gastrointestinal endoscopy after informed consent. Patients were excluded if they had taken Histamine2 receptor antagonists, proton pump inhibitors, antibiotics, bismuth compounds, or nonsteroidal antiinflammatory drugs (excluding low doses of aspirin) in the preceding 4 weeks.
Study Design Participants reported at 9 AM after a 12-hour fast. Intravenous sedation was given, and standard upper gastrointestinal endoscopy was performed to identify evidence of macroscopic abnormalities. Three biopsy specimens were obtained from the antral region within 2 cm of the pyloric ring, and three specimens were obtained from the fundus. A biopsy specimen from each site was used for rapid urease slide testing of H. pylori infection (CLO test; Delta West, Draper, Utah, USA), and the other two biopsy specimens were placed in 10% formalin and submitted for histologic examination. Before endoscopy, venous blood was drawn from each patient for serologic testing of H. pylori immunoglobulin (Ig) G antibodies. Sera were stored at ⫺20°C for analysis of IgG antibodies against H. pylori by using an enzyme-linked immunosorbent assay (ELIAS, Osceola, WI, USA) technique within 20 to 25 days. Simultaneously, saliva samples were also collected in sterile tubes for rapid urease activity. To prevent contamination of specimens taken from different sites, biopsy specimens were taken from each site with a fresh pair of sterile forceps. The forceps were wiped with alcohol on withdrawal from the endoscope to remove any organism that might have been present in the biopsy channel.
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Table 1. Treatment of Glaucoma Patients Treatment
No. Eyes
-blocker Dorzolamide Latanoprost -blocker, dorzolamide -blocker, latanoprost -blocker, pilocarpine Dorzolamide, latanoprost -blocker, pilocarpine and latanoprost None After trabeculectomy Blind
30 3 8 9 14 6 2 1 4 5
Endoscopes were sterilized between procedures according to standard guidelines.37
Biopsy and Saliva Urease Test Each biopsy specimen and saliva sample was placed in a tube containing 0.5 ml of 10% urea in deionized water to which had been added two drops of 1% phenol red as a pH indicator (CLO, Delta West). The biopsy specimen test was read at 5 minutes, 1 hour, 3 hours, and 24 hours and was regarded as positive if the indicator changed from yellow to red at any time. The saliva sample test was read at 5 minutes, 1 hour, and 3 hours.38,39
Histopathologic Analysis All specimens were stained with hematoxylin and eosin and with Cresyl fast violet stain, Giemsa stain, or both (for detection of H. pylori organisms). The presence of gastritis was classified in accordance with the Sydney system and included assessment of atrophy grade, chronicity, activity, and intestinal metaplasia on a scale of 0 (absent) to 3 (high), as previously reported.40,41 In particular, besides hematoxylin and eosin, intestinal metaplasia was evaluated with Alcian blue stain. An experienced pathologist assessed all specimens. The pathologist was unaware of the rapid urease test result and the clinical diagnosis.
Helicobacter pylori Serologic Analysis The H. pylori serologic analysis status was determined by using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The manufacturer’s recommended cut-off value of 10 units/ml for H. pylori IgG, validated in our laboratories, was used to define patient serologic analysis as positive or negative. The actual gold standard for the diagnosis of H. pylori infection was the detection of H. pylori organisms on microscopic analysis of mucosal biopsy specimens. Additional routine diagnostic criteria included positive rapid urease slide (biopsy specimens and saliva) and H. pylori IgG serologic analysis. For statistical analysis, chi-square test, Student’s t test, and nonparametric Mann–Whitney U test were performed where necessary.
Results Table 1 depicts the local antiglaucoma therapy used in our glaucoma patients. Mean age and gender ratios did not differ between glaucoma patients and anemic control participants (Table 2). In the glaucoma
Kountouras et al 䡠 Helicobacter pylori Infection and Glaucoma Table 2. Helicobacter pylori (H. pylori) Positively Detected by Urease Test in Saliva and Gastric Mucosa and by Histologic Analysis in Patients with Chronic Open-angle Glaucoma and Pseudoexfoliation Glaucoma and Anemic Control Participants Chronic Open-angle Glaucoma (Group a, n ⴝ 32)
Pseudoexfoliation Glaucoma (Group b, n ⴝ 9)
Controls (Group c, n ⴝ 30)
Age (yrs), median (range) Gender (men⬊women) Urease test saliva positive Urease test gastric mucosa positive
64 (45–70) 16⬊16 12 (37.5%) 23 (71.9%)
62 (51–70) 4⬊5 5 (55.6%) 7 (77.8%)
67.5 (44–70) 14⬊16 9 (30%) 14 (46.7%)
Histological presence of H. pylori
28 (87.5%)
8 (88.9%)
14 (46.7%)
Total H. pylori positive cases
28 (87.5%)
8 (88.9%)
14 (46.7%)
Significance Group a versus Group c NS NS NS P ⫽ 0.03; 95% Cl ⫽ 1.01–8.39 P ⫽ 0.0006; 95% Cl ⫽ 2.21–28.93 P ⫽ 0.0006; 95% Cl ⫽ 2.21–28.93 odds ratio; 8.00
Group b versus Group c NS NS NS NS P ⫽ 0.02; 95% Cl ⫽ 1.61–51.84 P ⫽ 0.02; 95% Cl ⫽ 1.61–51.84 odds ratio; 9.14
CI ⫽ confidence interval; NS ⫽ not significant.
patients, the mean visual acuity was 20/30, the mean cup-to-disc ratio was 0.5, the mean defect in the visual field was 3.19 dB (range, ⫺2.8 to 18.3 dB), and the mean short-term fluctuation was 2.23 dB (range, 0 – 4.1 dB). The prevalence of H. pylori infection was 87.5% in the COAG patients (28 of 32), 88.9% in the PEG patients (8 of 9), and 46.7% in the anemic control participants (14 of 30), as confirmed by the presence of H. pylori bacteria histologically (chi-square, 11.81; P ⫽ 0.0006; and chi-square, 5.01; P ⫽ 0.02, respectively; Table 2). The odds ratio for the association of H. pylori with the COAG subgroup was 8.00, and the 95% confidence interval ranged between 2.21 and 28.93. The odds ratio for the association of H. pylori with PEG subgroup was 9.14, and the 95% confidence interval ranged between 1.61 and 51.84. As shown in Table 2, H. pylori was detected: (1) in the saliva in 37.5% (12 of 32) of the COAG patients, in 55.6% (5 of 9) of the PEG patients, and 30% (9 of 30) of the anemic control participants (P ⬎ 0.05); and (2) in the gastric mucosa (antrum, corpus, or both) in 71.9% (23 of 32) of the COAG patients, 77.8% (7 of 9) of the PEG patients, and 46.7% (14 of 30) of the anemic control participants (P ⫽ 0.03; 95% confidence interval, 1.01– 8.39; P ⬎ 0.05, respectively) by urease test. Histologic presence of H. pylori was observed in 87.8% (36 of 41) of the glaucoma patients, including six of the glaucoma patients without positivity in the gastric mucosa urease test (P ⫽ 0.0002; 95% confidence interval, 2.53–26.74, for comparison with the anemic control value). Comparisons between the two glaucoma subgroups did not reveal any statistical difference. Increased serum levels of IgG anti-H. pylori (⬎10 U/ml) were observed in 68.3% (28 of 41) of the glaucoma patients, and in 30% (9 of 30) of the anemic control participants (P ⫽ 0.002; 95% confidence interval, 1.79 –13.7). When compared with the control values, the mean serum IgG anti-H. pylori level was also significantly higher in glaucoma patients (17.03 ⫾ 18.1 vs. 35.6 ⫾ 31.1 U/ml; P ⫽ 0.002). Increased serum levels of IgG anti-H. pylori were also noted in 68.8% (22 of 32) of the subgroup of COAG, and in 66.7% (6 of 9) of the subgroup of PEG. Table 3 shows the endoscopic evidence of esophagitis, gastritis, duodenitis, inefficiency of lower esophageal sphincter, hiatus hernia, peptic ulcer disease, or a combination noted in glaucoma patients and anemic control participants. When compared with the anemic control participants, glaucoma patients exhibited less often endoscopic normal appearance of the gastric mucosa (P ⫽ 0.01) and more often antral gastritis (P ⫽ 0.0004) or peptic ulcer disease (P ⫽ 0.01). Antral gastritis and peptic ulcer disease were confirmed histologically. Comparisons for endoscopic evidence of
esophagitis, duodenitis, inefficiency of lower esophageal sphincter, and hiatus hernia did not reveal statistical differences between glaucoma patients and anemic control participants (Table 3). There was no significant difference between the two glaucoma subgroups with regard to the endoscopic findings. The histologic grading of H. pylori infection (according to the Sydney system) in glaucoma patients versus the anemic control participants included atrophy grade, chronicity, activity, and intestinal metaplasia on a scale of 0 (absent) to 3 (high). Grade 3 was noted in 6 of 41 (14.6%) glaucoma patients and in none of the anemic control participants (chi-square, 4.795; P ⫽ 0.03), whereas grades 0, 1, and 2 did not differ significantly between glaucoma patients and anemic control participants. Comparisons for the histologic grading of H. pylori infection (Sydney system) in the two subgroups of glaucoma patients did not reveal any statistical difference. Topical administration of glaucoma agents was well tolerated in our glaucoma patients.
Discussion Since its original description by Warren and Marshall42 in 1983, H. pylori, a curved spiral gram-negative bacterium Table 3. Endoscopic Evidence of Esophagitis, Gastritis, Duodenitis, Inefficiency of Lower Esophageal Sphincter, Hiatus Hernia, and Peptic Ulcer Disease in Glaucoma Patients and Control Participants Glaucoma Patients Control Group P Value (n ⴝ 41) (n ⴝ 30) (Chi-square) Normal Esophagitis Antral gastritis Duodenitis Inefficiency of lower esophageal sphincter Hiatus hernia Peptic ulcer disease
1 (2.4%) 8 (19.5%) 38 (92.7%) 18 (43.9%) 17 (41.5%)
6 (20%) 2 (6.7%) 17 (56.7%) 13 (43.3%) 8 (26.7%)
3 (7.3%) 7 (17.1%)
2 (6.7%) 0 (0%)
P ⫽ 0.01 NS P ⫽ 0.0004 NS NS NS P ⫽ 0.01
NS ⫽ not significant.
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Ophthalmology Volume 108, Number 3, March 2001 that colonizes the gastric mucosa, has been associated with various upper gastrointestinal diseases43,44 and extradigestive disorders.24 –27 To the best of our knowledge, the present study suggests, for the first time, a link between H. pylori infection and glaucoma. In our cohort of Greek patients, 87.8% of the glaucoma patients exhibited histologically proven H. pylori infection, whereas the rate of infection was significantly lower in the anemic control group. The prevalence observed in our control group is similar to other published figures for Greeks, or other cohorts23 [Goritsas et al, Gut 45(suppl V):A108, 1999]. Our study has relied on histologic analysis for the documentation of H. pylori infection. Although culture is the theoretic gold standard for detection of the bacterium, it has been shown that there is an excellent correlation with histologic identification.45 Therefore, for most studies, mucosal biopsy and histologic examination of the specimen for the presence of H. pylori and gastritis is the actual gold standard for diagnosis of H. pylori infection.23,45 However, an upper gastrointestinal endoscopy is required to obtain specimens for histologic analysis or culture of H. pylori, and this method is costly and sometimes uncomfortable for patients. Therefore, for screening purposes, following up the efficacy of H. pylori treatment, or both, noninvasive tests, such as a breath test or serologic and saliva detection of antibodies to H. pylori, may be preferable. Urea breath tests are noninvasive and reliable but require an expensive isotope-ratio mass spectrometer or a nondispersible infrared spectrometer and produce a long half-life radioactive waste.46,47 Besides, this test requires fasting, false-negative results may occur if antibiotics have been used within the previous 4 weeks, and false-positive results can occur from urease present in the mouth.48 The presence of specific IgG antibodies against H. pylori can be shown by the relatively inexpensive and generally available enzyme-linked immunosorbent assay technique, although it requires invasive sampling of blood38,49 and, moreover, does not discriminate between current and old infections.48 Therefore, although our results did show a significant increase in serum IgG anti-H. pylori levels in glaucoma patients compared with control participants, the diagnostic significance of this test was limited because it could not distinguish between prior and current infections.48,50 More recently, saliva and dental plaque have been proposed as important reservoirs for H. pylori, based on the findings of polymerase chain reaction amplifications, cultures for H. pylori, and urease tests.39,49,51,52 Saliva urease testing appears to be useful for detection of H. pyloripositive patients with peptic ulcer disease.38 However, its diagnostic significance is limited by its low sensitivity rate, as seen in the present study. Recent data also suggest that gastric mucosa urease testing is also insensitive, especially in the elderly.53 Therefore, the endoscopic gastric mucosa histologic analysis appears to be the most reliable diagnostic procedure for H. pylori detection. However, for screening purposes alone, endoscopy is limited because it is costly and may lead to cardiopulmonary problems in elderly patients with chronic heart or lung disease.54
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H. pylori infection may increase the incidence or severity of glaucoma through the following mechanisms: 1. Promoting platelet and platelet–leukocyte aggregation.35 Platelet activation and aggregation have been proposed to play pathophysiologic roles in the development of glaucoma.11–18 2. Releasing large amounts of proinflammatory and vasoactive substances, such as cytokines (interleukin [IL]-1, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-␣, interferon-␥), eicosanoids (leukotrienes, prostaglandins), and acute phase proteins (fibrinogen, Creactive protein).25,27,30 In particular, increased endothelin-1 (a potent constrictor of arterioles and venules), nitric oxide, and nitric oxide synthase (iNOS) levels are associated with H. pylori infection.32,55 Endothelin-1–induced vasoconstriction of the anterior optic nerve vessels and nitric oxide modulation of vascular tone in the ophthalmic artery may produce glaucomatous damage.2,6 –10. 3. Stimulating mononuclear cells to produce a tissue factor-like procoagulant that converts fibrinogen into fibrin.56 4. Causing the development of cross mimicry between endothelial and H. pylori antigens.27 5. Producing reactive oxygen metabolites such as lipid peroxides.24,57 Reactive oxygen metabolites may be involved in the pathophysiology of glaucoma.58 The present study did not establish causality, because this requires us to show that eradication of H. pylori alters the course of glaucoma. The H. pylori and glaucoma association, reported herein, may be explained by an existence of a common (genetic?) factor that predispose to both H. pylori infection and glaucoma. An outgrowth of this alternative possibility would be that eradicating H. pylori infection would not necessarily have any effect on the development or progression of glaucoma. Genetic susceptibility to H. pylori infection has been reported in studies of monozygotic twins.23 However, future investigation for any potential detection of common genetic alterations causing susceptibility to both conditions is needed to elucidate this hypothesis. Chronic open-angle glaucoma and PEG are the two most common forms of open-angle glaucoma in Greece.59,60 It is well documented that these forms of glaucoma have a different pathogenesis, clinical course, and prognosis.61– 63 Our results show a high prevalence of H. pylori infection in both glaucomas. The significance of this finding is limited by the small number of PEG patients. Nevertheless, it is conceivable that both forms of glaucoma may share a common infectious link. The possibility of an infectious pathogenetic mechanism in PEG was implied in an epidemiologic study in Norway, where the prevalence of the condition was significantly more common in spouses.63 Further studies are required to elucidate this possibility. The glaucoma patients in the present study received only topical antiglaucoma medications. These drugs have no known effect on infectious manifestations or outcome. It is conceivable that a local carbonic anhydrase inhibitor, like dorzolamide, may influence the stomach pH and thus influ-
Kountouras et al 䡠 Helicobacter pylori Infection and Glaucoma ence the infectivity of H. pylori. In our small cohort of patients receiving dorzolamide, five of eight were positive for H. pylori (62.5%). Further research is required to clarify this point. In conclusion, an association between H. pylori infection and glaucoma in a Greek cohort has been found. Whether this association is causal or coincidental needs confirmation by other rigorously controlled epidemiologic studies. If a causal link between H. pylori infection and glaucoma is confirmed in the future, this may have a major impact on the pathophysiology and management of glaucoma. Acknowledgments: The authors thank F. Halkides, MD, for his expert help with some of the endoscopic procedures; P. Traianidis, MD, PhD, and M. Riga, MD, for referring some of the glaucoma patients included in this study; and A.G.P. Konstas, MD, PhD, for referring some of the glaucoma patients and for his constructive criticism of the manuscript.
14. 15.
16. 17. 18. 19. 20.
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cyclooxygenase 2 in Helicobacter pylori gastritis. Gastroenterology 1999;116:1319 –29. Markus HS, Mendall MA. Helicobacter pylori infection: a risk factor for ischaemic cerebrovascular disease and carotid atheroma. J Neurol Neurosurg Psychiatry 1998;64:104 –7. Panes J, Granger DN. Leukocyte-endothelial cell interactions: molecular mechanisms and implications in gastrointestinal disease. Gastroenterology 1998;114:1066 –90. Stewart WC. Clinical Practice of Glaucoma. Thorofare, NJ: SLACK, 1990;149 –96. British Society of Gastroenterology. Cleaning and disinfection of equipment for gastrointestinal flexible endoscopy: interim recommendations of a Working Party of the British Society of Gastroenterology. Gut 1988;29:1134 –51. Kountouras J, Boura P, Iliadis M, et al. Saliva urease test and serological surveillance of Helicobacter pylori infection during treatment of peptic ulcer disease. Hell J Gastroenterol 1996;9:67–71. Kountouras J. Detecting Helicobacter pylori. Diagnostic tests for Helicobacter pylori [letter]. Gut 1998;42:900 –1. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney system. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol 1996;20:1161– 81. Hatz RA, Rieder G, Stotle M, et al. Pattern of adhesion molecule expression on vascular endothelium in Helicobacter pylori-associated antral gastritis. Gastroenterology 1997;112: 1908 –19. Warren JR, Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis [letter]. Lancet 1983;1: 1273–5. Blaser MJ. Helicobacter pylori and the pathogenesis of gastroduodenal inflammation. J Infect Dis 1990;161:626 –33. NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. JAMA 1994;272:65–9. Weinstein WM. Gastritis and gastropathies. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal Disease: Pathology, Diagnosis, Management, 5th ed. Philadelphia: Saunders, 1993;545–71. Rauws EAJ, Royen EAV, Langenberg W, et al. 14C-urea breath test in C. pylori gastritis. Gut 1989;30:798 – 803. Koletzko S, Haisch M, Seeboth I, et al. Isotope-selective non-dispersive infrared spectrometry for detection of Helicobacter pylori infection with 13C-urea breath test. Lancet 1995; 345:961–2.
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48. Fennerty MB. Helicobacter pylori. Arch Intern Med 1994;154: 721–7. 49. Hopkins RJ, Morris JG Jr. Helicobacter pylori: the missing link in perspective. Am J Med 1994;97:265–77. 50. Meyer B, Werth B, Beglinger C, et al. Helicobacter pylori infection in healthy people: a dynamic process? Gut 1991;32: 347–50. 51. Banatvala N, Lopez CR, Owen R, et al. Helicobacter pylori in dental plaque [letter]. Lancet 1993;341:380. 52. Mapstone NP, Lynch DAF, Lewis FA, et al. Identification of Helicobacter pylori DNA in the mouths and stomachs of patients with gastritis using PCR. J Clin Pathol 1993;46: 540 –3. 53. Abdalla AM, Sordillo EM, Hanzely Z, et al. Insensitivity of the CLO test for H. pylori, especially in the elderly. Gastroenterology 1998;115:243– 4. 54. Young HS, Keeffe EB. Complications of gastrointestinal endoscopy. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal Disease. Philadelphia: Saunders, 1993;301–16. 55. Slomiany BL, Piotrowski J, Slomiany A. Up-regulation of endothelin-converting enzyme-1 in gastric mucosal inflammatory responses to Helicobacter pylori lipopolysaccharide. Biochem Biophys Res Commun 2000;267:801–5. 56. Miragliotta G, Del Prete R, Mosca A. Helicobacter pylori infection and coronary heart disease [letter]. Lancet 1994;334: 751. 57. Gotz JM, Thio JT, Verspaget HW, et al. Treatment of Helicobacter pylori infection favourably affects gastric mucosal superoxide dismutases. Gut 1997;40:591– 6. 58. McNamara J, Fridovich I. Human genetics. Did radicals strike Lou Gehring? Nature 1993;362:20 –1. 59. Konstas AGP, Tsatsos I, Kardasopoulos A, et al. Preoperative features of patients with exfoliation glaucoma and primary open-angle glaucoma. The AHEPA study. Acta Ophthalmol Scand 1998;76:208 –12. 60. Mylopoulos N. Pseudoexfoliative glaucoma. Epidemiological study in Northern Greece. New Trends in Ophthalmology 1993;3:25–30. 61. Konstas AGP, Mantziris DA, Stewart WC. Diurnal intraocular pressure in untreated exfoliation and primary open-angle glaucoma. Arch Ophthalmol 1997;115:182–5. 62. Konstas AGP, Jay JL, Marshall GE, Lee WR. Prevalence, diagnostic features, and response to trabeculectomy in exfoliation glaucoma. Ophthalmology 1993;100:619 –27. 63. Ringvold A. Epidemiology of the pseudo-exfoliation syndrome [review]. Acta Ophthalmol Scand 1999;77:371–5.
endothelium-dependent vascular regulation.2,4 –10 In addition, impaired ocular blood flow resulting from blood hyperviscosity (increased platelet aggregation on atheromatous plaques in the elderly)11–18 and cytokines may also be involved in the pathogenesis of this disease.19 –21 A theoretical relationship between glaucoma and H. pylori infection exists: (1) both are diseases of older adults in the developed world1,22,23; (2) chronic H. pylori infection may produce systemic disorders involving vascular tone resulting from the release of vasoactive and proinflammatory substances24 –33; and (3) H. pylori infection is associated with arteriosclerosis-induced increased platelet activation and aggregation.34,35 The aim of the study was to investigate whether H. pylori infection is associated with glaucoma.
Patients and Methods Participants Group 1, the control participants, consisted of 30 asymptomatic patients who were undergoing upper gastrointestinal endoscopy ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00598-4
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Ophthalmology Volume 108, Number 3, March 2001 for the investigation of mild anemia but in whom endoscopy was normal. Group 2, patients with documented glaucoma, consisted of 32 patients with chronic open-angle glaucoma (COAG) and 9 patients with pseudoexfoliation glaucoma (PEG). The glaucoma patients were included in the study using the following criteria: a history of intraocular pressure of 21 mmHg or more; typical optic nerve head changes including saucerization, rim thinning or notching in the inferior or superior temporal area of the optic nerve head, or total glaucomatous cupping36; and typical visual field loss including a paracentral, arcuate, or Seidel’s scotoma or a nasal step.36 Seven of our glaucoma patients were legally blind in one eye as a result of glaucoma. Exclusion criteria included all eye diseases other than glaucoma, diabetes mellitus, and a myopic refractive error exceeding ⫺8 diopters. All 71 participants were older than 45 years and younger than 70 years. The intraocular pressures were measured with a calibrated Goldmann applanation tonometer. All the glaucoma patients had prior experience with automated perimetry. The visual fields were assessed with the Octopus program G1 (Octopus 500EZ G1 programme, Luterzeag AG, Zurich, Switzerland) by the same perimetrists. None of the glaucoma patients received oral medications that could decrease intraocular pressure (e.g., carbonic anhydrase inhibitors). The control participants had recently been diagnosed with mild iron deficiency anemia. The diagnosis of anemia was based on history and the gastrointestinal investigation, and none of the participants had received any treatment (e.g., ferrous sulfate) before the diagnosis. The blood profile of the anemic control group was: mean hematocrit 34.4 ⫾ 1.3%, mean hemoglobin 10.3 ⫾ 0.5 g/100 ml, and mean serum ferritin levels 9.5 ⫾ 1.7 g/l (mean ⫾ standard deviation). The causes of iron deficiency anemia were: hemorrhoids in nine participants, diverticulosis in five participants, blood donation in four participants, genitalia bleeding in four female participants, and inadequate iron supply in two participants. The cause of the anemia could not be determined in six participants. All 71 patients (41 glaucoma patients, 30 anemic control participants) underwent elective diagnostic upper gastrointestinal endoscopy after informed consent. Patients were excluded if they had taken Histamine2 receptor antagonists, proton pump inhibitors, antibiotics, bismuth compounds, or nonsteroidal antiinflammatory drugs (excluding low doses of aspirin) in the preceding 4 weeks.
Study Design Participants reported at 9 AM after a 12-hour fast. Intravenous sedation was given, and standard upper gastrointestinal endoscopy was performed to identify evidence of macroscopic abnormalities. Three biopsy specimens were obtained from the antral region within 2 cm of the pyloric ring, and three specimens were obtained from the fundus. A biopsy specimen from each site was used for rapid urease slide testing of H. pylori infection (CLO test; Delta West, Draper, Utah, USA), and the other two biopsy specimens were placed in 10% formalin and submitted for histologic examination. Before endoscopy, venous blood was drawn from each patient for serologic testing of H. pylori immunoglobulin (Ig) G antibodies. Sera were stored at ⫺20°C for analysis of IgG antibodies against H. pylori by using an enzyme-linked immunosorbent assay (ELIAS, Osceola, WI, USA) technique within 20 to 25 days. Simultaneously, saliva samples were also collected in sterile tubes for rapid urease activity. To prevent contamination of specimens taken from different sites, biopsy specimens were taken from each site with a fresh pair of sterile forceps. The forceps were wiped with alcohol on withdrawal from the endoscope to remove any organism that might have been present in the biopsy channel.
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Table 1. Treatment of Glaucoma Patients Treatment
No. Eyes
-blocker Dorzolamide Latanoprost -blocker, dorzolamide -blocker, latanoprost -blocker, pilocarpine Dorzolamide, latanoprost -blocker, pilocarpine and latanoprost None After trabeculectomy Blind
30 3 8 9 14 6 2 1 4 5
Endoscopes were sterilized between procedures according to standard guidelines.37
Biopsy and Saliva Urease Test Each biopsy specimen and saliva sample was placed in a tube containing 0.5 ml of 10% urea in deionized water to which had been added two drops of 1% phenol red as a pH indicator (CLO, Delta West). The biopsy specimen test was read at 5 minutes, 1 hour, 3 hours, and 24 hours and was regarded as positive if the indicator changed from yellow to red at any time. The saliva sample test was read at 5 minutes, 1 hour, and 3 hours.38,39
Histopathologic Analysis All specimens were stained with hematoxylin and eosin and with Cresyl fast violet stain, Giemsa stain, or both (for detection of H. pylori organisms). The presence of gastritis was classified in accordance with the Sydney system and included assessment of atrophy grade, chronicity, activity, and intestinal metaplasia on a scale of 0 (absent) to 3 (high), as previously reported.40,41 In particular, besides hematoxylin and eosin, intestinal metaplasia was evaluated with Alcian blue stain. An experienced pathologist assessed all specimens. The pathologist was unaware of the rapid urease test result and the clinical diagnosis.
Helicobacter pylori Serologic Analysis The H. pylori serologic analysis status was determined by using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The manufacturer’s recommended cut-off value of 10 units/ml for H. pylori IgG, validated in our laboratories, was used to define patient serologic analysis as positive or negative. The actual gold standard for the diagnosis of H. pylori infection was the detection of H. pylori organisms on microscopic analysis of mucosal biopsy specimens. Additional routine diagnostic criteria included positive rapid urease slide (biopsy specimens and saliva) and H. pylori IgG serologic analysis. For statistical analysis, chi-square test, Student’s t test, and nonparametric Mann–Whitney U test were performed where necessary.
Results Table 1 depicts the local antiglaucoma therapy used in our glaucoma patients. Mean age and gender ratios did not differ between glaucoma patients and anemic control participants (Table 2). In the glaucoma
Kountouras et al 䡠 Helicobacter pylori Infection and Glaucoma Table 2. Helicobacter pylori (H. pylori) Positively Detected by Urease Test in Saliva and Gastric Mucosa and by Histologic Analysis in Patients with Chronic Open-angle Glaucoma and Pseudoexfoliation Glaucoma and Anemic Control Participants Chronic Open-angle Glaucoma (Group a, n ⴝ 32)
Pseudoexfoliation Glaucoma (Group b, n ⴝ 9)
Controls (Group c, n ⴝ 30)
Age (yrs), median (range) Gender (men⬊women) Urease test saliva positive Urease test gastric mucosa positive
64 (45–70) 16⬊16 12 (37.5%) 23 (71.9%)
62 (51–70) 4⬊5 5 (55.6%) 7 (77.8%)
67.5 (44–70) 14⬊16 9 (30%) 14 (46.7%)
Histological presence of H. pylori
28 (87.5%)
8 (88.9%)
14 (46.7%)
Total H. pylori positive cases
28 (87.5%)
8 (88.9%)
14 (46.7%)
Significance Group a versus Group c NS NS NS P ⫽ 0.03; 95% Cl ⫽ 1.01–8.39 P ⫽ 0.0006; 95% Cl ⫽ 2.21–28.93 P ⫽ 0.0006; 95% Cl ⫽ 2.21–28.93 odds ratio; 8.00
Group b versus Group c NS NS NS NS P ⫽ 0.02; 95% Cl ⫽ 1.61–51.84 P ⫽ 0.02; 95% Cl ⫽ 1.61–51.84 odds ratio; 9.14
CI ⫽ confidence interval; NS ⫽ not significant.
patients, the mean visual acuity was 20/30, the mean cup-to-disc ratio was 0.5, the mean defect in the visual field was 3.19 dB (range, ⫺2.8 to 18.3 dB), and the mean short-term fluctuation was 2.23 dB (range, 0 – 4.1 dB). The prevalence of H. pylori infection was 87.5% in the COAG patients (28 of 32), 88.9% in the PEG patients (8 of 9), and 46.7% in the anemic control participants (14 of 30), as confirmed by the presence of H. pylori bacteria histologically (chi-square, 11.81; P ⫽ 0.0006; and chi-square, 5.01; P ⫽ 0.02, respectively; Table 2). The odds ratio for the association of H. pylori with the COAG subgroup was 8.00, and the 95% confidence interval ranged between 2.21 and 28.93. The odds ratio for the association of H. pylori with PEG subgroup was 9.14, and the 95% confidence interval ranged between 1.61 and 51.84. As shown in Table 2, H. pylori was detected: (1) in the saliva in 37.5% (12 of 32) of the COAG patients, in 55.6% (5 of 9) of the PEG patients, and 30% (9 of 30) of the anemic control participants (P ⬎ 0.05); and (2) in the gastric mucosa (antrum, corpus, or both) in 71.9% (23 of 32) of the COAG patients, 77.8% (7 of 9) of the PEG patients, and 46.7% (14 of 30) of the anemic control participants (P ⫽ 0.03; 95% confidence interval, 1.01– 8.39; P ⬎ 0.05, respectively) by urease test. Histologic presence of H. pylori was observed in 87.8% (36 of 41) of the glaucoma patients, including six of the glaucoma patients without positivity in the gastric mucosa urease test (P ⫽ 0.0002; 95% confidence interval, 2.53–26.74, for comparison with the anemic control value). Comparisons between the two glaucoma subgroups did not reveal any statistical difference. Increased serum levels of IgG anti-H. pylori (⬎10 U/ml) were observed in 68.3% (28 of 41) of the glaucoma patients, and in 30% (9 of 30) of the anemic control participants (P ⫽ 0.002; 95% confidence interval, 1.79 –13.7). When compared with the control values, the mean serum IgG anti-H. pylori level was also significantly higher in glaucoma patients (17.03 ⫾ 18.1 vs. 35.6 ⫾ 31.1 U/ml; P ⫽ 0.002). Increased serum levels of IgG anti-H. pylori were also noted in 68.8% (22 of 32) of the subgroup of COAG, and in 66.7% (6 of 9) of the subgroup of PEG. Table 3 shows the endoscopic evidence of esophagitis, gastritis, duodenitis, inefficiency of lower esophageal sphincter, hiatus hernia, peptic ulcer disease, or a combination noted in glaucoma patients and anemic control participants. When compared with the anemic control participants, glaucoma patients exhibited less often endoscopic normal appearance of the gastric mucosa (P ⫽ 0.01) and more often antral gastritis (P ⫽ 0.0004) or peptic ulcer disease (P ⫽ 0.01). Antral gastritis and peptic ulcer disease were confirmed histologically. Comparisons for endoscopic evidence of
esophagitis, duodenitis, inefficiency of lower esophageal sphincter, and hiatus hernia did not reveal statistical differences between glaucoma patients and anemic control participants (Table 3). There was no significant difference between the two glaucoma subgroups with regard to the endoscopic findings. The histologic grading of H. pylori infection (according to the Sydney system) in glaucoma patients versus the anemic control participants included atrophy grade, chronicity, activity, and intestinal metaplasia on a scale of 0 (absent) to 3 (high). Grade 3 was noted in 6 of 41 (14.6%) glaucoma patients and in none of the anemic control participants (chi-square, 4.795; P ⫽ 0.03), whereas grades 0, 1, and 2 did not differ significantly between glaucoma patients and anemic control participants. Comparisons for the histologic grading of H. pylori infection (Sydney system) in the two subgroups of glaucoma patients did not reveal any statistical difference. Topical administration of glaucoma agents was well tolerated in our glaucoma patients.
Discussion Since its original description by Warren and Marshall42 in 1983, H. pylori, a curved spiral gram-negative bacterium Table 3. Endoscopic Evidence of Esophagitis, Gastritis, Duodenitis, Inefficiency of Lower Esophageal Sphincter, Hiatus Hernia, and Peptic Ulcer Disease in Glaucoma Patients and Control Participants Glaucoma Patients Control Group P Value (n ⴝ 41) (n ⴝ 30) (Chi-square) Normal Esophagitis Antral gastritis Duodenitis Inefficiency of lower esophageal sphincter Hiatus hernia Peptic ulcer disease
1 (2.4%) 8 (19.5%) 38 (92.7%) 18 (43.9%) 17 (41.5%)
6 (20%) 2 (6.7%) 17 (56.7%) 13 (43.3%) 8 (26.7%)
3 (7.3%) 7 (17.1%)
2 (6.7%) 0 (0%)
P ⫽ 0.01 NS P ⫽ 0.0004 NS NS NS P ⫽ 0.01
NS ⫽ not significant.
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Ophthalmology Volume 108, Number 3, March 2001 that colonizes the gastric mucosa, has been associated with various upper gastrointestinal diseases43,44 and extradigestive disorders.24 –27 To the best of our knowledge, the present study suggests, for the first time, a link between H. pylori infection and glaucoma. In our cohort of Greek patients, 87.8% of the glaucoma patients exhibited histologically proven H. pylori infection, whereas the rate of infection was significantly lower in the anemic control group. The prevalence observed in our control group is similar to other published figures for Greeks, or other cohorts23 [Goritsas et al, Gut 45(suppl V):A108, 1999]. Our study has relied on histologic analysis for the documentation of H. pylori infection. Although culture is the theoretic gold standard for detection of the bacterium, it has been shown that there is an excellent correlation with histologic identification.45 Therefore, for most studies, mucosal biopsy and histologic examination of the specimen for the presence of H. pylori and gastritis is the actual gold standard for diagnosis of H. pylori infection.23,45 However, an upper gastrointestinal endoscopy is required to obtain specimens for histologic analysis or culture of H. pylori, and this method is costly and sometimes uncomfortable for patients. Therefore, for screening purposes, following up the efficacy of H. pylori treatment, or both, noninvasive tests, such as a breath test or serologic and saliva detection of antibodies to H. pylori, may be preferable. Urea breath tests are noninvasive and reliable but require an expensive isotope-ratio mass spectrometer or a nondispersible infrared spectrometer and produce a long half-life radioactive waste.46,47 Besides, this test requires fasting, false-negative results may occur if antibiotics have been used within the previous 4 weeks, and false-positive results can occur from urease present in the mouth.48 The presence of specific IgG antibodies against H. pylori can be shown by the relatively inexpensive and generally available enzyme-linked immunosorbent assay technique, although it requires invasive sampling of blood38,49 and, moreover, does not discriminate between current and old infections.48 Therefore, although our results did show a significant increase in serum IgG anti-H. pylori levels in glaucoma patients compared with control participants, the diagnostic significance of this test was limited because it could not distinguish between prior and current infections.48,50 More recently, saliva and dental plaque have been proposed as important reservoirs for H. pylori, based on the findings of polymerase chain reaction amplifications, cultures for H. pylori, and urease tests.39,49,51,52 Saliva urease testing appears to be useful for detection of H. pyloripositive patients with peptic ulcer disease.38 However, its diagnostic significance is limited by its low sensitivity rate, as seen in the present study. Recent data also suggest that gastric mucosa urease testing is also insensitive, especially in the elderly.53 Therefore, the endoscopic gastric mucosa histologic analysis appears to be the most reliable diagnostic procedure for H. pylori detection. However, for screening purposes alone, endoscopy is limited because it is costly and may lead to cardiopulmonary problems in elderly patients with chronic heart or lung disease.54
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H. pylori infection may increase the incidence or severity of glaucoma through the following mechanisms: 1. Promoting platelet and platelet–leukocyte aggregation.35 Platelet activation and aggregation have been proposed to play pathophysiologic roles in the development of glaucoma.11–18 2. Releasing large amounts of proinflammatory and vasoactive substances, such as cytokines (interleukin [IL]-1, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-␣, interferon-␥), eicosanoids (leukotrienes, prostaglandins), and acute phase proteins (fibrinogen, Creactive protein).25,27,30 In particular, increased endothelin-1 (a potent constrictor of arterioles and venules), nitric oxide, and nitric oxide synthase (iNOS) levels are associated with H. pylori infection.32,55 Endothelin-1–induced vasoconstriction of the anterior optic nerve vessels and nitric oxide modulation of vascular tone in the ophthalmic artery may produce glaucomatous damage.2,6 –10. 3. Stimulating mononuclear cells to produce a tissue factor-like procoagulant that converts fibrinogen into fibrin.56 4. Causing the development of cross mimicry between endothelial and H. pylori antigens.27 5. Producing reactive oxygen metabolites such as lipid peroxides.24,57 Reactive oxygen metabolites may be involved in the pathophysiology of glaucoma.58 The present study did not establish causality, because this requires us to show that eradication of H. pylori alters the course of glaucoma. The H. pylori and glaucoma association, reported herein, may be explained by an existence of a common (genetic?) factor that predispose to both H. pylori infection and glaucoma. An outgrowth of this alternative possibility would be that eradicating H. pylori infection would not necessarily have any effect on the development or progression of glaucoma. Genetic susceptibility to H. pylori infection has been reported in studies of monozygotic twins.23 However, future investigation for any potential detection of common genetic alterations causing susceptibility to both conditions is needed to elucidate this hypothesis. Chronic open-angle glaucoma and PEG are the two most common forms of open-angle glaucoma in Greece.59,60 It is well documented that these forms of glaucoma have a different pathogenesis, clinical course, and prognosis.61– 63 Our results show a high prevalence of H. pylori infection in both glaucomas. The significance of this finding is limited by the small number of PEG patients. Nevertheless, it is conceivable that both forms of glaucoma may share a common infectious link. The possibility of an infectious pathogenetic mechanism in PEG was implied in an epidemiologic study in Norway, where the prevalence of the condition was significantly more common in spouses.63 Further studies are required to elucidate this possibility. The glaucoma patients in the present study received only topical antiglaucoma medications. These drugs have no known effect on infectious manifestations or outcome. It is conceivable that a local carbonic anhydrase inhibitor, like dorzolamide, may influence the stomach pH and thus influ-
Kountouras et al 䡠 Helicobacter pylori Infection and Glaucoma ence the infectivity of H. pylori. In our small cohort of patients receiving dorzolamide, five of eight were positive for H. pylori (62.5%). Further research is required to clarify this point. In conclusion, an association between H. pylori infection and glaucoma in a Greek cohort has been found. Whether this association is causal or coincidental needs confirmation by other rigorously controlled epidemiologic studies. If a causal link between H. pylori infection and glaucoma is confirmed in the future, this may have a major impact on the pathophysiology and management of glaucoma. Acknowledgments: The authors thank F. Halkides, MD, for his expert help with some of the endoscopic procedures; P. Traianidis, MD, PhD, and M. Riga, MD, for referring some of the glaucoma patients included in this study; and A.G.P. Konstas, MD, PhD, for referring some of the glaucoma patients and for his constructive criticism of the manuscript.
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