ORIGINAL STUDY
Latent Infections as a Risk Factor for Posttrabeculectomy Bleb Failure Ernest V. Boiko, MD, DSc, Alexei L. Pozniak, MD, DSc, Dmitrii I. Iakushev, MD, Dmitrii S. Maltsev, MD, PhD, Alexei A. Suetov, MD, and Irina V. Nuralova, MD, PhD
Purpose: To investigate latent conjunctival Chlamydia trachomatis (CT) and Bacteroides fragilis (BF) infections as potential risk factors for posttrabeculectomy bleb failure. Patients and Methods: This retrospective observational study included 50 primary open-angle glaucoma eyes of 50 patients who were submitted to trabeculectomy without cytostatics from September 2010 to June 2011 and were followed up for at least a year. Preoperatively, conjunctival scrapings were taken and their specimens subjected to polymerase chain reaction, direct fluorescent assay and cell culture testing for CT, and culture for BF on blood agar medium. Serum CT-specific IgG and IgA and tear interleukin (IL)-1b and IL-8 concentrations were measured with enzyme-linked immunosorbent assay. We defined bleb failure as intraocular pressure >21 mm Hg with antiglaucoma medications, resulting from reduced bleb filtration capacity due to bleb fibrosis, fistula obstruction, flattened bleb, or encapsulated bleb, and no earlier than 2 weeks after surgery. At the time of the reintervention, a scleroconjunctival biopsy was obtained for histopathology (including direct fluorescent assay testing for CT). Eyes were divided into a failure group and a nonfailure group, depending on whether they developed bleb failure (required reintervention) or not within a follow-up year. Results: In the failure group (n = 18), the frequencies of detection of CT and BF in conjunctival specimens were 27.8% and 66.7%, respectively, versus 0% and 9.4% in the nonfailure group (n = 32). CT and BF were detected in 11.1% and 11.1%, respectively, of scleroconjunctival biopsies. IgG and IgA seropositivity to CT was found in 66.7% and 33.3%, respectively, of the failure group patients, versus 9.4% and 0% of the nonfailure group patients. Tear IL-1b and IL-8 levels were markedly elevated in the failure group (468.83 ± 80.43 and 107.89 ± 15.11 pg/mL, respectively) versus the nonfailure group (22.34 ± 5.43 and 9.34 ± 2.83 pg/mL, respectively). Conclusion: Being a contributor to low-grade conjunctival inflammation, latent conjunctival CT, and BF infections in primary open-angle glaucoma patients represent risk factors for posttrabeculectomy bleb failure. Key Words: glaucoma, trabeculectomy, bleb failure, C. trachomatis, B. fragilis
(J Glaucoma 2016;25:306–311)
T
he success of trabeculectomy for primary open-angle glaucoma (POAG) is often hampered by bleb failure,1–3 with failure rates ranging from 22% to 74%.4–6 This process is a manifestation of marked reparative regeneration and depends on the level of inflammatory and proliferative activity.3 Therefore, inflammatory changes in the conjunctiva (particularly, chronic conjunctivitis) are considered as a risk factor for the failure of filtration surgery.7,8 Chronic conjunctivitis can be caused by such microorganisms as C. trachomatis (CT)9 and anaerobes,10 whose key role in chronic inflammation has been established recently. CT is of a rather high prevalence in different populations (3.4% to 12%)11–13 and tends to damage the ocular anterior segment,14 whereas B. fragilis (BF) is an anaerobic microorganism and a component of the resident human flora15 capable of being activated in damaged tissues, especially when associated with other microorganisms.15 Therefore, these agents are of interest as potential infectious etiologic factors of low-grade chronic conjunctival inflammation, with the latter possibly resulting in scar-related failure of trabeculectomy. The degree of conjunctival infiltration with inflammatory cells and inflammatory cytokine levels in tears16 can reflect the degree of activity of conjunctival inflammation; hence, studying these indices along with detection of the infection may facilitate the search of potential risk factors for bleb scarring. Because studies have shown the key role of interleukin (IL)-1b and IL-8 in the pathogenesis of CTspecific diseases14,17 and BF-induced inflammation,18 studying the levels of these very ILs in tears and their relationship with chronic infection and posttrabeculectomy bleb failure is deemed to be worthwhile. To our knowledge, this is the first study that focuses on the detection of these infectious agents in the bleb tissue of posttrabeculectomy glaucoma patients.
MATERIALS AND METHODS This was a single-center, nonrandomized, retrospective observational study.
Patients Received for publication February 11, 2014; accepted November 27, 2014. From the Department of Ophthalmology, Kirov Military Medical Academy, St Petersburg, Russia. Disclosure: The authors declare no conflict of interest. Reprints: Ernest V. Boiko, MD, DSc, Department of Ophthalmology, Military Medical Academy, 5 Klinicheskaya Street, St. Petersburg, 194044, Russia (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/IJG.0000000000000212
The study adhered to the tenets of the Declaration of Helsinki and was approved by Ethics Committee of Military Medical Academy (St Petersburg, Russia). Informed consent was obtained from all study patients. The study included 50 POAG eyes of 50 patients (aged 55 to 83 y) who were submitted to trabeculectomy without cytostatics from September 2010 to June 2011 and were followed up for at least a year. Exclusion criteria included a recent history (r2 y) of or current conjunctivitis; any history of past surgery or ocular trauma in the POAG eye;
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endocrine diseases; systemic diseases of connective tissue; or a history of recent (r12 mo) or current use of antibiotic, anti-inflammatory, cytostatic, or hormonal agents. In all study eyes, primary interventions (trabeculectomies) were performed by the same surgeon using the same surgical technique. Both after the initial surgery and after the reintervention surgery, the patients were treated with antimicrobial and anti-inflammatory monotherapy, tobramycin and dexamethasone ophthalmic suspension (Tobradex; Alcon-Couvreur, Puurs, Belgium). At the baseline and at 12-month visits, all study patients had comprehensive ophthalmic examination, applanation tonometry, gonioscopy, and static perimetry with the 24-2 Swedish interactive test algorithm [SITA] standard test (Humphrey Field Analyzer II; Zeiss-Humphrey Systems, Dublin, CA), and had the content of their aintiglaucoma agents (active substances and preservatives) evaluated. Follow-up visits were scheduled at day 1; weeks 1 and 2; months 2, 4, 6, 8, 10, and 12. We defined bleb failure as an inability to control the intraocular pressure (IOP > 21 mm Hg) with antiglaucoma medications of at least 2 classes on 3 consecutive follow-up visits, resulting from reduced bleb filtration capacity, providing the capacity reduction occurs due to bleb fibrosis, fistula obstruction (excluding internal sclerotomy blockage by iris, blood, etc.), flattened bleb, or encapsulated bleb, and no earlier than 2 weeks after surgery. Defined in this way, bleb failure was used as the indication for reintervention. Reintervention was performed by the same surgeon who performed the initial intervention, and its extent varied from bleb revision to retrabeculectomy in the adjacent quadrant. Study eyes were divided into a failure group and a nonfailure group, involving those that underwent reintervention and those that did not require it within a follow-up year, respectively. The anti-inflammatory treatment regimen did not vary in the failure and nonfailure groups.
Sample Collection In all study eyes, before initial surgical intervention, conjunctival scrapings were taken to detect CT and BF. Conjunctival scrape smears were subjected to direct fluorescent assay (DFA) for identification of CT. The excess material from scraping was placed into Transport Medium with Mucolytic Agent (Amplisens, Moscow, Russia) for polymerase chain reaction (PCR) and universal transport medium (Copan, Murrieta, CA) for culture examination. Scraping material was plated onto anaerobic blood agar (Thermo Scientific, Waltham, MA) to detect BF. A patient was considered positive for CT infection if he/she was found to be positive by all the methods.
Tear Sample Collection Ten-microliter tear samples were taken from the lower tear meniscus using sterile glass pipettes and kept at 701C until used for cytokine analysis.
Blood Sample Collection Preoperatively, 10 mL venous blood samples were taken from the cubital vein of each patient to determine the antibody levels to the infectious agents under study. After blood clot formation, serum was separated and kept at 701C until used for antibody titer determination. Copyright
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Latent Infections as a Risk Factor for Bleb Failure
DFA Smears were fixed in 70% cold methanol and incubated in the presence of ChlamySlide fluorescein isothiocyanate-conjugated antibodies (LABDiagnostica, Moscow, Russia) to CT cell wall lipopolysaccharide in a moisture chamber, according to the manufacturer’s instructions. After being washed in phosphate buffer saline and dried, smears were subjected to microscopy. Evaluation was performed if the amount of epithelial cells in a scrape sample was at least 50. A sample was considered positive if >10 loci of specific fluorescence were identified on the slide.
PCR Reagent kits—DNA-sorb-B Nucleic Acid Extraction kit (AmpliSens) and Chlamydia trachomatis-screen-titerFRT PCR kit (AmpliSens)—were used to perform PCR, following the manufacturer’s instructions. Real-time PCR was performed on a rotary analyzer Rotor-Gene 6000 (Corbett Research, Sydney, Australia).
Cell Culture Universal transport medium of 0.3 mL that contained the conjunctival scraping samples was introduced into cycloheximide-treated McCoy cell culture. At 72 hours postinoculation with specimens under study, the culture was fixed in 70% cold methanol. DFA testing was performed according to the manufacturer’s instructions.
Cytokine Assay To evaluate the activity of inflammatory process, IL-1b and IL-8 cytokine levels were quantitated with enzyme-linked immunosorbent assay (ELISA) kits, Interleukin-1b-IFA-Best (Vector-Best, Novosibirsk, Russia) and Interleukin-8-IFA-Best (Vector-Best), with sensitivities of 1 and 2 pg/mL, respectively, following the manufacturer’s instructions. Before ELISA analysis, each 10-mL tear sample was supplemented with 100 mL of phosphate buffer saline containing 0.5% fetal bovine serum.
ELISA Serum CT-specific IgG and IgA were measured with ELISA kits, ImmunoComb Chlamydia trachomatis IgG (Orgenics, Yavne, Israel) and Chlamydia trachomatis monovalent IgA (Orgenics), following the manufacturer’s instructions. A titer of IgG or IgA to CT of 1:8 or higher was considered diagnostically significant.
Histopathology At the time of the reintervention, a scleroconjunctival biopsy was obtained for histopathologic examination. Paraffin-embedded sections were stained with hematoxylin and eosin. Adjacent sections were subjected to DFA following the manufacturer’s protocol.
Statistical Analysis Nonparametric data analysis was performed with Statistica for Windows 6.0 software (Statsoft, Tulsa, OK). Mann-Whitney U tests and Fisher exact test were used to compare anatomic and demographic parameters of the groups. Wilcoxon signed-rank test was used for pairwise comparison between changes in IOP, MD index, and cytokine levels. Statistical significance was taken as P < 0.05.
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TABLE 1. Baseline Characteristics of Patients With (n = 18) and Without (n = 32) Bleb Failure
Factors Age (mean ± SD) (y) Sex (male/female) Refraction (D) Glaucoma severity (MD) (dB) IOP (mm Hg) IL-1b (pg/mL) IL-8 (pg/mL)
72.22 ± 7.31 2/16 0.33 ± 1.38 14.86 ± 3.58 29.17 ± 2.57 468.83 ± 80.43 107.89 ± 15.11
Nonfailure Group (n = 32) 70.53 ± 8.84 6/26 0.46 ± 1.07 12.56 ± 3.78
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and IL-8 levels were not statistically significantly different among the types of topical antiglaucoma therapy (Fig. 1).
CT-specific Antibodies
Total (N = 50) Failure Group (n = 18)
P 0.746 0.484 0.527 0.072
27.81 ± 1.9 0.143 22.34 ± 5.43 0.000 9.34 ± 2.83 0.000
IL indicates interleukin; IOP indicates intraocular pressure.
In the failure group, increased IgA and IgG titers to CT were found in 6 and 12 patients, respectively. Five of these 6 cases of active infection had diagnostically significant IgA titers, and corresponded to the cases with identification of CT in conjunctival scrapings. In 4 of these 5 cases, this corresponded to increased IgG titers to CT, thus pointing to reactivation of chronic infection. In nonfailure patients found to be positive by at least one but not by all of the methods, the antibody titers did not exceed the level of diagnostic significance. In addition, in the failure group, no patient was found to have increased IgA antibody titers, and 3 patients were found to have diagnostically significant IgG antibody titers.
Histopathology RESULTS Baseline Characteristics and Follow-up After primary intervention, in the failure and nonfailure groups, mean IOP was 21.61 ± 2.64 and 20 ± 2.1 mm Hg (P = 0.076), respectively. Eighteen patients required reintervention after a mean period of 5.28 ± 1.88 months (range, 2 wk to 8 mo) (Table 1). There was no difference in pretrabeculectomy antiglaucoma therapy between study groups (P > 0.05); patients were treated with different combinations of timolol maleat, latanoprost, or dorzolamide (all these contain benzalkonium chloride). Posttrabeculectomy, antiglaucoma therapy (either mono or combination therapy) was used mostly in patients of the failure group, whereas in the nonfailure group, 4 patients had monotherapy with timolol maleat or latanoprost.
Identification of Infectious Agents CT and BF were detected significantly more often (P < 0.01) in the failure group. Five patients (27.8%) of this group were positive for CT by all of the 3 methods (ie, DFA, PCR, and culture). None of the patients of the nonfailure group were positive for CT by all of the 3 methods (Table 2). The frequency of detection of BF was higher in the failure group than in the nonfailure group (66.7% vs. 9.4%).
Tear IL-1b and IL-8 Levels Tear IL-1b and IL-8 levels were higher, although not statistically significantly so, in the failure group compared with the nonfailure group (P < 0.001) (Table 1). Tear IL-1b
The histopathology of biopsy material revealed localized indurated fibers of the scleral tissue, reduced number of cell elements, and isolated microcysts in the episclera. Marked mixed inflammatory infiltration was observed in the conjunctiva and subconjunctival tissue (Fig. 2A). In 2 subjects, cells of BF were revealed in superficial scleral layers (Fig. 2B). The histopathology of biopsy material from patients with laboratory proved CT infection revealed extracellular CT elementary bodies in the conjunctival and scleral tissue (Figs. 2C, D), without simultaneous detection of BF by light microscopy, in 2 subjects (40% of the subjects found to be positive by all the methods).
DISCUSSION Filtration surgery remains the gold standard for treatment of medically uncontrolled POAG2,19 and understanding the mechanisms of filtration failure is important for improvement of the success rate of this procedure.20 Part of the reason for failures of trabeculectomies performed without antimetabolites is bleb failure (as defined in our study), with failure rates ranging from 22% to 74%,4–6 despite adherence to the intervention protocol.4–6 In our study, trabeculectomy failure rate (as determined by percentage of patients that required reintervention within a year) was 36%, most likely, due to the use of trabeculectomy without cytostatics. In addition, in the failure group, the degree of conjunctival inflammation (reflected in tear IL-1b and IL-8 levels) and CT + BF detection rate based on examination of conjunctival scraping were reliably higher than those in the nonfailure group. Furthermore, the infectious agents were detected directly in the bleb tissues (ie, the conjunctival tissue and superficial scleral layers)—
TABLE 2. Results of Detection of Chlamydia trachomatis and Bacteroides fragilis in Patients With and Without Bleb Failure
Groups
Infectious Agents
DFA
PCR
Culture
Blood Agar
Failure
Chlamydia trachomatis Bacteroides fragilis Chlamydia trachomatis Bacteroides fragilis
10 (55.6) — 2 (6.25) —
5 (27.8) — 0 —
6 (33.3) — 0 —
— 12 (66.7) — 3 (9.38)
Nonfailure
The values represents n (%). DFA indicates direct fluorescent assay; PCR, polymerase chain reaction.
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FIGURE 1. Baseline tear interleukin (IL)-1b and IL-8 levels in the eyes with bleb failure versus type of topical antiglaucoma therapy. The boundaries of each box indicate the 25th and 75th percentiles, the whiskers indicate the minimum and maximum values.
the tissues, the proliferative changes in which result in bleb failure. Therefore, one may believe that these pathogenic microorganisms caused increased tear IL-1b and IL-8 levels by supporting low-grade chronic conjunctival
Latent Infections as a Risk Factor for Bleb Failure
inflammation, the latter being an established risk factor for bleb failure. Because the study groups were not different in other risk factors (in particular, age, IOP level, severity of the disease, earlier antiglaucoma therapy, and type of preservative contained in therapeutic agents), these factors had no influence on the results of the study. A number of species of bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Haemophilus influenzae, Streptococcus spp.) can also cause conjunctival inflammation, which, however, is usually symptomatic (conjunctivitis), and these infectious agents rarely cause chronic infection. Moreover, aminoglycoside antibiotics used postoperatively in this study have no marked influence on CT and BF, but suppress the more typical flora (Staphylococcus spp., Streptococcus spp.) to minimize its influence on postoperative inflammatory process. Presence of CT in the conjunctiva was detected in 10% of the study eyes, this index value being close to those revealed by screening studies (3.4% to 12%).11–13 This corresponds to the previously reported absence of increased antibody titers to CT in POAG patients21 and demonstrates that CT infection is not necessarily directly related to development of glaucoma. Nevertheless, the reliably higher percentage of CT-positive patients (27.8%) in the failure
FIGURE 2. Histology sections of bleb tissue of the eyes that failed trabeculectomy. A, Conjuntival infiltration with inflammatory cells (arrowheads). B, Bacteroides fragilis cells (arrowheads) in superficial scleral layers [A and B: hematoxylin and eosin; (A) scale bar = 50 mm; (B) scale bar = 10 mm]. C, Chlamydia trachomatis (CT) elementary bodies (arrowheads) in the conjunctival tissue. D, CT elementary bodies (arrowheads) in superficial scleral layers (C and D: direct fluorescent assay, scale bar = 10 mm).
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group indicates that patients with this infection have an increased risk of scarring and severity of clinical course of POAG. In the failure group, increased percentage of CTpositive patients was also proved by the increased IgA and IgG titers to CT, evidencing the chronicity of the course of infection. BF is a component of the resident human flora, and this partially explains why its detection frequency (30%) was higher than that of CT.15 However, BF detected with high frequency in the failure group, when associated with CT, possibly exhibited pathogenic properties by supporting and contributing to inflammatory process and scarring. Considering the findings, preoperative CT and BF tests might be appropriate for glaucoma patients to evaluate the risk of bleb scarring. If the results of these tests were positive, adequate therapy for the infections would be required, including possibly systemic use of proper antibiotics, such as azithromycin and metronidazole; the former has proved to be efficient in the treatment of ocular chlamydia infection,22 whereas the latter is active against BF.23 However, this issue needs further study. Nevertheless, topical fluoroquinolones may be used postoperatively instead of narrower spectrum antibiotics, aminoglycosides, or chloramphenicol, already now. We cannot completely rule out the possibility that persistent infections may participate in the natural course of POAG. If so, this might be related in some way to high level of proinflammatory cytokines in the aqueous humor.24,25 Some authors have already hypothesized that glaucoma is related to chronic infections.26,27 In glaucoma patients, it is likely that not just 1 specific latent infectious agent is associated with the development and progress of the disease, causing the obturation of conventional aqueous humor outflow pathways due to low-grade chronic persistent inflammation,28 and this issue requires further investigation. The increased IL-1b and IL-8 levels (found in our study in tears of failure group patients) are related to inflammation and scarring process in the CT-infected tissues; this occurs also in trachoma, the latter being characterized by apparent conjunctival scarring.14 Moreover, BF apparently stimulates IL-8 production and moderately stimulates that of IL-1b.18 Regarding the results of our study, the increased cytokine levels may be accounted for conjunctival inflammation due to persistent infection. Undoubtedly, after trabeculectomy, as after any surgical intervention, inflammation can be caused by trauma involving breakdown of the periscleral and scleral tissues and ocular drainage system. However, patients who had undergone the same surgical protocol had different outcomes with time. Because (1) only baseline cytokine levels were taken into account; and (2) at the baseline, patients received antiglaucoma eye drop treatment which did not differ significantly between the groups, the influence of local therapy on IL-1b and IL-8 levels may be neglected. Moreover, tear IL-1b and IL-8 levels were not statistically significantly different for any of the 3 types of topical antiglaucoma therapy (Fig. 1). The presence of conjunctival inflammation in patients with bleb failure was confirmed also by the histopathology which revealed inflammatory infiltrations in biopsy materials taken at the time of the reintervention. The fact that the inflammation was low grade does not rule out its pathogenetic importance27 and explains why its clinical manifestations, when combined with postoperative changes, do not attract the attention of the attending ophthalmologist. Dry eye syndrome correlates with
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glaucoma and can influence the intensity of conjunctival inflammation and IL levels.29 We did not assess the degrees of dry eye syndrome, and this may be a limitation of the study. Other limitations of the study include the use of trabeculectomy without antimetabolites, which is no longer a standard of care (however, the outcomes associated with the use of these drugs in patients with chronic conjunctival infections are difficult to predict and will require a separate investigation), and the retrospective nonblind design. In conclusion, the results of this study give evidence for the important role of CT-induced and BF-induced latent chronic inflammatory process in the development of excessive bleb scarring in fistulizing operations for glaucoma. REFERENCES 1. Barton K, Budenz DL, Khaw PT, et al. Glaucoma filtration surgery using amniotic membrane transplantation. Invest Ophthalmol Vis Sci. 2001;42:1762–1768. 2. Dahan E, Ben Simon GJ, Lafuma A. Comparison of trabeculectomy and Ex-PRESS implantation in fellow eyes of the same patient: a prospective, randomised study. Eye (Lond). 2012;26:703–710. 3. Cvenkel B, Kopitar AN, Ihan A. Inflammatory molecules in aqueous humour and on ocular surface and glaucoma surgery outcome. Mediators Inflamm. 2010;2010:939602. 4. The Fluorouracil Filtering Surgery Study Group. Five-year follow-up of the Fluorouracil Filtering Surgery Study. Am J Ophthalmol. 1996;121:349–366. 5. Gedde SJ, Schiffman JC, Feuer WJ, et al. Tube versus Trabeculectomy Study Group. Treatment outcomes in the Tube Versus Trabeculectomy (TVT) study after five years of follow-up. Am J Ophthalmol. 2012;153:789–803. 6. Landers J, Martin K, Sarkies N, et al. A twenty-year follow-up study of trabeculectomy: risk factors and outcomes. Ophthalmology. 2012;119:694–702. 7. Helin M, Ro¨nkko¨ S, Puustja¨rvi T, et al. Conjunctival inflammatory cells and their predictive role for deep sclerectomy in primary open-angle glaucoma and exfoliation glaucoma. J Glaucoma. 2011;20:172–178. 8. Shaarawy T, Sherwood M, Hitchings R, et al. Glaucoma Volume 2: Surgical Management. Philadelphia, PA: Elsevier Limited; 2009. 9. Hu VH, Holland MJ, Burton MJ. Trachoma: protective and pathogenic ocular immune responses to Chlamydia trachomatis. PLoS Negl Trop Dis. 2013;7:e2020. 10. Winkelhoff AJ, Abbas F, Pavicic MJ, et al. Chronic conjunctivitis caused by oral anaerobic germs. Ned Tijdschr Geneeskd. 1991;135:2489–2491. 11. Carne CA, Gibbs J, Delaney A, et al. Prevalence, clinical features and quantification of genital non-viral infections. Int J STD AIDS. 2013;24:273–277. 12. Mascellino MT, Ciardi MR, Oliva A, et al. Chlamydia trachomatis detection in a population of asymptomatic and symptomatic women: correlation with the presence of serological markers for this infection. New Microbiol. 2008;31:249–256. 13. Lockington D, MacDonald R, King S, et al. Multiplex PCR testing requires a robust multi-disciplinary strategy to effectively manage identified cases of chlamydial conjunctivitis. Scott Med J. 2013;58:77–82. 14. Skwor TA, Atik B, Kandel RP, et al. Role of secreted conjunctival mucosal cytokine and chemokine proteins in different stages of trachomatous disease. PLoS Negl Trop Dis. 2008;2:e264. 15. Wexler HM. Bacteroides: the good, the bad, and the nittygritty. Clin Microbiol Rev. 2007;20:593–621. 16. Malvitte L, Montange T, Vejux A, et al. Measurement of inflammatory cytokines by multicytokine assay in tears of patients with glaucoma topically treated with chronic drugs. Br J Ophthalmol. 2007;91:29–32.
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17. Daponte A, Pournaras S, Deligeoroglou E, et al. Serum interleukin-1b, interleukin-8 and anti-heat shock 60 Chlamydia trachomatis antibodies as markers of ectopic pregnancy. J Reprod Immunol. 2012;93:102–108. 18. Stappers MH, Janssen NA, Oosting M, et al. A role for TLR1, TLR2 and NOD2 in cytokine induction by Bacteroides fragilis. Cytokine. 2012;60:861–869. 19. Lukowski ZL, Min J, Beattie AR, et al. Prevention of ocular scarring after glaucoma filtering surgery using the monoclonal antibody LT1009 (Sonepcizumab) in a rabbit model. J Glaucoma. 2013;22:145–151. 20. Coleman AL. Advances in glaucoma treatment and management: surgery. Invest Ophthalmol Vis Sci. 2012;53: 2491–2494. 21. Yuki K, Kimura I, Shiba D, et al. Elevated serum immunoglobulin G titers against Chlamydia pneumoniae in primary open-angle glaucoma patients without systemic disease. J Glaucoma. 2010;19:535–539. 22. Malamos P, Georgalas I, Rallis K, et al. Evaluation of singledose azithromycin versus standard azithromycin/doxycycline treatment and clinical assessment of regression course in patients with adult inclusion conjunctivitis. Curr Eye Res. 2013;38:1198–1206.
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23. Schaumann R, Funke M, Janssen E, et al. In vitro activities of clindamycin, imipenem, metronidazole, and piperacillin-tazobactam against susceptible and resistant isolates of Bacteroides fragilis evaluated by kill kinetics. Antimicrob Agents Chemother. 2012;56:3413–3416. 24. Inoue T, Kawaji T, Inatani M, et al. Simultaneous increases in multiple proinflammatory cytokines in the aqueous humor in pseudophakic glaucomatous eyes. J Cataract Refract Surg. 2012;38:1389–1397. 25. Chua J, Vania M, Cheung CM, et al. Expression profile of inflammatory cytokines in aqueous from glaucomatous eyes. Mol Vis. 2012;18:431–438. 26. Izzotti A, Sacca` SC, Bagnis A, et al. Glaucoma and Helicobacter pylori infection: correlations and controversies. Br J Ophthalmol. 2009;93:1420–1427. 27. Vohra R, Tsai JC, Kolko M. The role of inflammation in the pathogenesis of glaucoma. Surv Ophthalmol. 2013;58:311–320. 28. Volkov VV. On differences in the pathogenesis, clinical course, treatment, and prevention of glaucoma and ischemic optic neuropathy. Vestn Oftalmol. 2010;126:3–14. 29. Schmier JK, Covert DW. Characteristics of respondents with glaucoma and dry eye in a national panel survey. Clin Ophthalmol. 2009;3:645–650.
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Latent Infections as a Risk Factor for Posttrabeculectomy Bleb Failure Ernest V. Boiko, MD, DSc, Alexei L. Pozniak, MD, DSc, Dmitrii I. Iakushev, MD, Dmitrii S. Maltsev, MD, PhD, Alexei A. Suetov, MD, and Irina V. Nuralova, MD, PhD
Purpose: To investigate latent conjunctival Chlamydia trachomatis (CT) and Bacteroides fragilis (BF) infections as potential risk factors for posttrabeculectomy bleb failure. Patients and Methods: This retrospective observational study included 50 primary open-angle glaucoma eyes of 50 patients who were submitted to trabeculectomy without cytostatics from September 2010 to June 2011 and were followed up for at least a year. Preoperatively, conjunctival scrapings were taken and their specimens subjected to polymerase chain reaction, direct fluorescent assay and cell culture testing for CT, and culture for BF on blood agar medium. Serum CT-specific IgG and IgA and tear interleukin (IL)-1b and IL-8 concentrations were measured with enzyme-linked immunosorbent assay. We defined bleb failure as intraocular pressure >21 mm Hg with antiglaucoma medications, resulting from reduced bleb filtration capacity due to bleb fibrosis, fistula obstruction, flattened bleb, or encapsulated bleb, and no earlier than 2 weeks after surgery. At the time of the reintervention, a scleroconjunctival biopsy was obtained for histopathology (including direct fluorescent assay testing for CT). Eyes were divided into a failure group and a nonfailure group, depending on whether they developed bleb failure (required reintervention) or not within a follow-up year. Results: In the failure group (n = 18), the frequencies of detection of CT and BF in conjunctival specimens were 27.8% and 66.7%, respectively, versus 0% and 9.4% in the nonfailure group (n = 32). CT and BF were detected in 11.1% and 11.1%, respectively, of scleroconjunctival biopsies. IgG and IgA seropositivity to CT was found in 66.7% and 33.3%, respectively, of the failure group patients, versus 9.4% and 0% of the nonfailure group patients. Tear IL-1b and IL-8 levels were markedly elevated in the failure group (468.83 ± 80.43 and 107.89 ± 15.11 pg/mL, respectively) versus the nonfailure group (22.34 ± 5.43 and 9.34 ± 2.83 pg/mL, respectively). Conclusion: Being a contributor to low-grade conjunctival inflammation, latent conjunctival CT, and BF infections in primary open-angle glaucoma patients represent risk factors for posttrabeculectomy bleb failure. Key Words: glaucoma, trabeculectomy, bleb failure, C. trachomatis, B. fragilis
(J Glaucoma 2016;25:306–311)
T
he success of trabeculectomy for primary open-angle glaucoma (POAG) is often hampered by bleb failure,1–3 with failure rates ranging from 22% to 74%.4–6 This process is a manifestation of marked reparative regeneration and depends on the level of inflammatory and proliferative activity.3 Therefore, inflammatory changes in the conjunctiva (particularly, chronic conjunctivitis) are considered as a risk factor for the failure of filtration surgery.7,8 Chronic conjunctivitis can be caused by such microorganisms as C. trachomatis (CT)9 and anaerobes,10 whose key role in chronic inflammation has been established recently. CT is of a rather high prevalence in different populations (3.4% to 12%)11–13 and tends to damage the ocular anterior segment,14 whereas B. fragilis (BF) is an anaerobic microorganism and a component of the resident human flora15 capable of being activated in damaged tissues, especially when associated with other microorganisms.15 Therefore, these agents are of interest as potential infectious etiologic factors of low-grade chronic conjunctival inflammation, with the latter possibly resulting in scar-related failure of trabeculectomy. The degree of conjunctival infiltration with inflammatory cells and inflammatory cytokine levels in tears16 can reflect the degree of activity of conjunctival inflammation; hence, studying these indices along with detection of the infection may facilitate the search of potential risk factors for bleb scarring. Because studies have shown the key role of interleukin (IL)-1b and IL-8 in the pathogenesis of CTspecific diseases14,17 and BF-induced inflammation,18 studying the levels of these very ILs in tears and their relationship with chronic infection and posttrabeculectomy bleb failure is deemed to be worthwhile. To our knowledge, this is the first study that focuses on the detection of these infectious agents in the bleb tissue of posttrabeculectomy glaucoma patients.
MATERIALS AND METHODS This was a single-center, nonrandomized, retrospective observational study.
Patients Received for publication February 11, 2014; accepted November 27, 2014. From the Department of Ophthalmology, Kirov Military Medical Academy, St Petersburg, Russia. Disclosure: The authors declare no conflict of interest. Reprints: Ernest V. Boiko, MD, DSc, Department of Ophthalmology, Military Medical Academy, 5 Klinicheskaya Street, St. Petersburg, 194044, Russia (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/IJG.0000000000000212
The study adhered to the tenets of the Declaration of Helsinki and was approved by Ethics Committee of Military Medical Academy (St Petersburg, Russia). Informed consent was obtained from all study patients. The study included 50 POAG eyes of 50 patients (aged 55 to 83 y) who were submitted to trabeculectomy without cytostatics from September 2010 to June 2011 and were followed up for at least a year. Exclusion criteria included a recent history (r2 y) of or current conjunctivitis; any history of past surgery or ocular trauma in the POAG eye;
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endocrine diseases; systemic diseases of connective tissue; or a history of recent (r12 mo) or current use of antibiotic, anti-inflammatory, cytostatic, or hormonal agents. In all study eyes, primary interventions (trabeculectomies) were performed by the same surgeon using the same surgical technique. Both after the initial surgery and after the reintervention surgery, the patients were treated with antimicrobial and anti-inflammatory monotherapy, tobramycin and dexamethasone ophthalmic suspension (Tobradex; Alcon-Couvreur, Puurs, Belgium). At the baseline and at 12-month visits, all study patients had comprehensive ophthalmic examination, applanation tonometry, gonioscopy, and static perimetry with the 24-2 Swedish interactive test algorithm [SITA] standard test (Humphrey Field Analyzer II; Zeiss-Humphrey Systems, Dublin, CA), and had the content of their aintiglaucoma agents (active substances and preservatives) evaluated. Follow-up visits were scheduled at day 1; weeks 1 and 2; months 2, 4, 6, 8, 10, and 12. We defined bleb failure as an inability to control the intraocular pressure (IOP > 21 mm Hg) with antiglaucoma medications of at least 2 classes on 3 consecutive follow-up visits, resulting from reduced bleb filtration capacity, providing the capacity reduction occurs due to bleb fibrosis, fistula obstruction (excluding internal sclerotomy blockage by iris, blood, etc.), flattened bleb, or encapsulated bleb, and no earlier than 2 weeks after surgery. Defined in this way, bleb failure was used as the indication for reintervention. Reintervention was performed by the same surgeon who performed the initial intervention, and its extent varied from bleb revision to retrabeculectomy in the adjacent quadrant. Study eyes were divided into a failure group and a nonfailure group, involving those that underwent reintervention and those that did not require it within a follow-up year, respectively. The anti-inflammatory treatment regimen did not vary in the failure and nonfailure groups.
Sample Collection In all study eyes, before initial surgical intervention, conjunctival scrapings were taken to detect CT and BF. Conjunctival scrape smears were subjected to direct fluorescent assay (DFA) for identification of CT. The excess material from scraping was placed into Transport Medium with Mucolytic Agent (Amplisens, Moscow, Russia) for polymerase chain reaction (PCR) and universal transport medium (Copan, Murrieta, CA) for culture examination. Scraping material was plated onto anaerobic blood agar (Thermo Scientific, Waltham, MA) to detect BF. A patient was considered positive for CT infection if he/she was found to be positive by all the methods.
Tear Sample Collection Ten-microliter tear samples were taken from the lower tear meniscus using sterile glass pipettes and kept at 701C until used for cytokine analysis.
Blood Sample Collection Preoperatively, 10 mL venous blood samples were taken from the cubital vein of each patient to determine the antibody levels to the infectious agents under study. After blood clot formation, serum was separated and kept at 701C until used for antibody titer determination. Copyright
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Latent Infections as a Risk Factor for Bleb Failure
DFA Smears were fixed in 70% cold methanol and incubated in the presence of ChlamySlide fluorescein isothiocyanate-conjugated antibodies (LABDiagnostica, Moscow, Russia) to CT cell wall lipopolysaccharide in a moisture chamber, according to the manufacturer’s instructions. After being washed in phosphate buffer saline and dried, smears were subjected to microscopy. Evaluation was performed if the amount of epithelial cells in a scrape sample was at least 50. A sample was considered positive if >10 loci of specific fluorescence were identified on the slide.
PCR Reagent kits—DNA-sorb-B Nucleic Acid Extraction kit (AmpliSens) and Chlamydia trachomatis-screen-titerFRT PCR kit (AmpliSens)—were used to perform PCR, following the manufacturer’s instructions. Real-time PCR was performed on a rotary analyzer Rotor-Gene 6000 (Corbett Research, Sydney, Australia).
Cell Culture Universal transport medium of 0.3 mL that contained the conjunctival scraping samples was introduced into cycloheximide-treated McCoy cell culture. At 72 hours postinoculation with specimens under study, the culture was fixed in 70% cold methanol. DFA testing was performed according to the manufacturer’s instructions.
Cytokine Assay To evaluate the activity of inflammatory process, IL-1b and IL-8 cytokine levels were quantitated with enzyme-linked immunosorbent assay (ELISA) kits, Interleukin-1b-IFA-Best (Vector-Best, Novosibirsk, Russia) and Interleukin-8-IFA-Best (Vector-Best), with sensitivities of 1 and 2 pg/mL, respectively, following the manufacturer’s instructions. Before ELISA analysis, each 10-mL tear sample was supplemented with 100 mL of phosphate buffer saline containing 0.5% fetal bovine serum.
ELISA Serum CT-specific IgG and IgA were measured with ELISA kits, ImmunoComb Chlamydia trachomatis IgG (Orgenics, Yavne, Israel) and Chlamydia trachomatis monovalent IgA (Orgenics), following the manufacturer’s instructions. A titer of IgG or IgA to CT of 1:8 or higher was considered diagnostically significant.
Histopathology At the time of the reintervention, a scleroconjunctival biopsy was obtained for histopathologic examination. Paraffin-embedded sections were stained with hematoxylin and eosin. Adjacent sections were subjected to DFA following the manufacturer’s protocol.
Statistical Analysis Nonparametric data analysis was performed with Statistica for Windows 6.0 software (Statsoft, Tulsa, OK). Mann-Whitney U tests and Fisher exact test were used to compare anatomic and demographic parameters of the groups. Wilcoxon signed-rank test was used for pairwise comparison between changes in IOP, MD index, and cytokine levels. Statistical significance was taken as P < 0.05.
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TABLE 1. Baseline Characteristics of Patients With (n = 18) and Without (n = 32) Bleb Failure
Factors Age (mean ± SD) (y) Sex (male/female) Refraction (D) Glaucoma severity (MD) (dB) IOP (mm Hg) IL-1b (pg/mL) IL-8 (pg/mL)
72.22 ± 7.31 2/16 0.33 ± 1.38 14.86 ± 3.58 29.17 ± 2.57 468.83 ± 80.43 107.89 ± 15.11
Nonfailure Group (n = 32) 70.53 ± 8.84 6/26 0.46 ± 1.07 12.56 ± 3.78
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and IL-8 levels were not statistically significantly different among the types of topical antiglaucoma therapy (Fig. 1).
CT-specific Antibodies
Total (N = 50) Failure Group (n = 18)
P 0.746 0.484 0.527 0.072
27.81 ± 1.9 0.143 22.34 ± 5.43 0.000 9.34 ± 2.83 0.000
IL indicates interleukin; IOP indicates intraocular pressure.
In the failure group, increased IgA and IgG titers to CT were found in 6 and 12 patients, respectively. Five of these 6 cases of active infection had diagnostically significant IgA titers, and corresponded to the cases with identification of CT in conjunctival scrapings. In 4 of these 5 cases, this corresponded to increased IgG titers to CT, thus pointing to reactivation of chronic infection. In nonfailure patients found to be positive by at least one but not by all of the methods, the antibody titers did not exceed the level of diagnostic significance. In addition, in the failure group, no patient was found to have increased IgA antibody titers, and 3 patients were found to have diagnostically significant IgG antibody titers.
Histopathology RESULTS Baseline Characteristics and Follow-up After primary intervention, in the failure and nonfailure groups, mean IOP was 21.61 ± 2.64 and 20 ± 2.1 mm Hg (P = 0.076), respectively. Eighteen patients required reintervention after a mean period of 5.28 ± 1.88 months (range, 2 wk to 8 mo) (Table 1). There was no difference in pretrabeculectomy antiglaucoma therapy between study groups (P > 0.05); patients were treated with different combinations of timolol maleat, latanoprost, or dorzolamide (all these contain benzalkonium chloride). Posttrabeculectomy, antiglaucoma therapy (either mono or combination therapy) was used mostly in patients of the failure group, whereas in the nonfailure group, 4 patients had monotherapy with timolol maleat or latanoprost.
Identification of Infectious Agents CT and BF were detected significantly more often (P < 0.01) in the failure group. Five patients (27.8%) of this group were positive for CT by all of the 3 methods (ie, DFA, PCR, and culture). None of the patients of the nonfailure group were positive for CT by all of the 3 methods (Table 2). The frequency of detection of BF was higher in the failure group than in the nonfailure group (66.7% vs. 9.4%).
Tear IL-1b and IL-8 Levels Tear IL-1b and IL-8 levels were higher, although not statistically significantly so, in the failure group compared with the nonfailure group (P < 0.001) (Table 1). Tear IL-1b
The histopathology of biopsy material revealed localized indurated fibers of the scleral tissue, reduced number of cell elements, and isolated microcysts in the episclera. Marked mixed inflammatory infiltration was observed in the conjunctiva and subconjunctival tissue (Fig. 2A). In 2 subjects, cells of BF were revealed in superficial scleral layers (Fig. 2B). The histopathology of biopsy material from patients with laboratory proved CT infection revealed extracellular CT elementary bodies in the conjunctival and scleral tissue (Figs. 2C, D), without simultaneous detection of BF by light microscopy, in 2 subjects (40% of the subjects found to be positive by all the methods).
DISCUSSION Filtration surgery remains the gold standard for treatment of medically uncontrolled POAG2,19 and understanding the mechanisms of filtration failure is important for improvement of the success rate of this procedure.20 Part of the reason for failures of trabeculectomies performed without antimetabolites is bleb failure (as defined in our study), with failure rates ranging from 22% to 74%,4–6 despite adherence to the intervention protocol.4–6 In our study, trabeculectomy failure rate (as determined by percentage of patients that required reintervention within a year) was 36%, most likely, due to the use of trabeculectomy without cytostatics. In addition, in the failure group, the degree of conjunctival inflammation (reflected in tear IL-1b and IL-8 levels) and CT + BF detection rate based on examination of conjunctival scraping were reliably higher than those in the nonfailure group. Furthermore, the infectious agents were detected directly in the bleb tissues (ie, the conjunctival tissue and superficial scleral layers)—
TABLE 2. Results of Detection of Chlamydia trachomatis and Bacteroides fragilis in Patients With and Without Bleb Failure
Groups
Infectious Agents
DFA
PCR
Culture
Blood Agar
Failure
Chlamydia trachomatis Bacteroides fragilis Chlamydia trachomatis Bacteroides fragilis
10 (55.6) — 2 (6.25) —
5 (27.8) — 0 —
6 (33.3) — 0 —
— 12 (66.7) — 3 (9.38)
Nonfailure
The values represents n (%). DFA indicates direct fluorescent assay; PCR, polymerase chain reaction.
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FIGURE 1. Baseline tear interleukin (IL)-1b and IL-8 levels in the eyes with bleb failure versus type of topical antiglaucoma therapy. The boundaries of each box indicate the 25th and 75th percentiles, the whiskers indicate the minimum and maximum values.
the tissues, the proliferative changes in which result in bleb failure. Therefore, one may believe that these pathogenic microorganisms caused increased tear IL-1b and IL-8 levels by supporting low-grade chronic conjunctival
Latent Infections as a Risk Factor for Bleb Failure
inflammation, the latter being an established risk factor for bleb failure. Because the study groups were not different in other risk factors (in particular, age, IOP level, severity of the disease, earlier antiglaucoma therapy, and type of preservative contained in therapeutic agents), these factors had no influence on the results of the study. A number of species of bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Haemophilus influenzae, Streptococcus spp.) can also cause conjunctival inflammation, which, however, is usually symptomatic (conjunctivitis), and these infectious agents rarely cause chronic infection. Moreover, aminoglycoside antibiotics used postoperatively in this study have no marked influence on CT and BF, but suppress the more typical flora (Staphylococcus spp., Streptococcus spp.) to minimize its influence on postoperative inflammatory process. Presence of CT in the conjunctiva was detected in 10% of the study eyes, this index value being close to those revealed by screening studies (3.4% to 12%).11–13 This corresponds to the previously reported absence of increased antibody titers to CT in POAG patients21 and demonstrates that CT infection is not necessarily directly related to development of glaucoma. Nevertheless, the reliably higher percentage of CT-positive patients (27.8%) in the failure
FIGURE 2. Histology sections of bleb tissue of the eyes that failed trabeculectomy. A, Conjuntival infiltration with inflammatory cells (arrowheads). B, Bacteroides fragilis cells (arrowheads) in superficial scleral layers [A and B: hematoxylin and eosin; (A) scale bar = 50 mm; (B) scale bar = 10 mm]. C, Chlamydia trachomatis (CT) elementary bodies (arrowheads) in the conjunctival tissue. D, CT elementary bodies (arrowheads) in superficial scleral layers (C and D: direct fluorescent assay, scale bar = 10 mm).
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group indicates that patients with this infection have an increased risk of scarring and severity of clinical course of POAG. In the failure group, increased percentage of CTpositive patients was also proved by the increased IgA and IgG titers to CT, evidencing the chronicity of the course of infection. BF is a component of the resident human flora, and this partially explains why its detection frequency (30%) was higher than that of CT.15 However, BF detected with high frequency in the failure group, when associated with CT, possibly exhibited pathogenic properties by supporting and contributing to inflammatory process and scarring. Considering the findings, preoperative CT and BF tests might be appropriate for glaucoma patients to evaluate the risk of bleb scarring. If the results of these tests were positive, adequate therapy for the infections would be required, including possibly systemic use of proper antibiotics, such as azithromycin and metronidazole; the former has proved to be efficient in the treatment of ocular chlamydia infection,22 whereas the latter is active against BF.23 However, this issue needs further study. Nevertheless, topical fluoroquinolones may be used postoperatively instead of narrower spectrum antibiotics, aminoglycosides, or chloramphenicol, already now. We cannot completely rule out the possibility that persistent infections may participate in the natural course of POAG. If so, this might be related in some way to high level of proinflammatory cytokines in the aqueous humor.24,25 Some authors have already hypothesized that glaucoma is related to chronic infections.26,27 In glaucoma patients, it is likely that not just 1 specific latent infectious agent is associated with the development and progress of the disease, causing the obturation of conventional aqueous humor outflow pathways due to low-grade chronic persistent inflammation,28 and this issue requires further investigation. The increased IL-1b and IL-8 levels (found in our study in tears of failure group patients) are related to inflammation and scarring process in the CT-infected tissues; this occurs also in trachoma, the latter being characterized by apparent conjunctival scarring.14 Moreover, BF apparently stimulates IL-8 production and moderately stimulates that of IL-1b.18 Regarding the results of our study, the increased cytokine levels may be accounted for conjunctival inflammation due to persistent infection. Undoubtedly, after trabeculectomy, as after any surgical intervention, inflammation can be caused by trauma involving breakdown of the periscleral and scleral tissues and ocular drainage system. However, patients who had undergone the same surgical protocol had different outcomes with time. Because (1) only baseline cytokine levels were taken into account; and (2) at the baseline, patients received antiglaucoma eye drop treatment which did not differ significantly between the groups, the influence of local therapy on IL-1b and IL-8 levels may be neglected. Moreover, tear IL-1b and IL-8 levels were not statistically significantly different for any of the 3 types of topical antiglaucoma therapy (Fig. 1). The presence of conjunctival inflammation in patients with bleb failure was confirmed also by the histopathology which revealed inflammatory infiltrations in biopsy materials taken at the time of the reintervention. The fact that the inflammation was low grade does not rule out its pathogenetic importance27 and explains why its clinical manifestations, when combined with postoperative changes, do not attract the attention of the attending ophthalmologist. Dry eye syndrome correlates with
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glaucoma and can influence the intensity of conjunctival inflammation and IL levels.29 We did not assess the degrees of dry eye syndrome, and this may be a limitation of the study. Other limitations of the study include the use of trabeculectomy without antimetabolites, which is no longer a standard of care (however, the outcomes associated with the use of these drugs in patients with chronic conjunctival infections are difficult to predict and will require a separate investigation), and the retrospective nonblind design. In conclusion, the results of this study give evidence for the important role of CT-induced and BF-induced latent chronic inflammatory process in the development of excessive bleb scarring in fistulizing operations for glaucoma. REFERENCES 1. Barton K, Budenz DL, Khaw PT, et al. Glaucoma filtration surgery using amniotic membrane transplantation. Invest Ophthalmol Vis Sci. 2001;42:1762–1768. 2. Dahan E, Ben Simon GJ, Lafuma A. Comparison of trabeculectomy and Ex-PRESS implantation in fellow eyes of the same patient: a prospective, randomised study. Eye (Lond). 2012;26:703–710. 3. Cvenkel B, Kopitar AN, Ihan A. Inflammatory molecules in aqueous humour and on ocular surface and glaucoma surgery outcome. Mediators Inflamm. 2010;2010:939602. 4. The Fluorouracil Filtering Surgery Study Group. Five-year follow-up of the Fluorouracil Filtering Surgery Study. Am J Ophthalmol. 1996;121:349–366. 5. Gedde SJ, Schiffman JC, Feuer WJ, et al. Tube versus Trabeculectomy Study Group. Treatment outcomes in the Tube Versus Trabeculectomy (TVT) study after five years of follow-up. Am J Ophthalmol. 2012;153:789–803. 6. Landers J, Martin K, Sarkies N, et al. A twenty-year follow-up study of trabeculectomy: risk factors and outcomes. Ophthalmology. 2012;119:694–702. 7. Helin M, Ro¨nkko¨ S, Puustja¨rvi T, et al. Conjunctival inflammatory cells and their predictive role for deep sclerectomy in primary open-angle glaucoma and exfoliation glaucoma. J Glaucoma. 2011;20:172–178. 8. Shaarawy T, Sherwood M, Hitchings R, et al. Glaucoma Volume 2: Surgical Management. Philadelphia, PA: Elsevier Limited; 2009. 9. Hu VH, Holland MJ, Burton MJ. Trachoma: protective and pathogenic ocular immune responses to Chlamydia trachomatis. PLoS Negl Trop Dis. 2013;7:e2020. 10. Winkelhoff AJ, Abbas F, Pavicic MJ, et al. Chronic conjunctivitis caused by oral anaerobic germs. Ned Tijdschr Geneeskd. 1991;135:2489–2491. 11. Carne CA, Gibbs J, Delaney A, et al. Prevalence, clinical features and quantification of genital non-viral infections. Int J STD AIDS. 2013;24:273–277. 12. Mascellino MT, Ciardi MR, Oliva A, et al. Chlamydia trachomatis detection in a population of asymptomatic and symptomatic women: correlation with the presence of serological markers for this infection. New Microbiol. 2008;31:249–256. 13. Lockington D, MacDonald R, King S, et al. Multiplex PCR testing requires a robust multi-disciplinary strategy to effectively manage identified cases of chlamydial conjunctivitis. Scott Med J. 2013;58:77–82. 14. Skwor TA, Atik B, Kandel RP, et al. Role of secreted conjunctival mucosal cytokine and chemokine proteins in different stages of trachomatous disease. PLoS Negl Trop Dis. 2008;2:e264. 15. Wexler HM. Bacteroides: the good, the bad, and the nittygritty. Clin Microbiol Rev. 2007;20:593–621. 16. Malvitte L, Montange T, Vejux A, et al. Measurement of inflammatory cytokines by multicytokine assay in tears of patients with glaucoma topically treated with chronic drugs. Br J Ophthalmol. 2007;91:29–32.
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17. Daponte A, Pournaras S, Deligeoroglou E, et al. Serum interleukin-1b, interleukin-8 and anti-heat shock 60 Chlamydia trachomatis antibodies as markers of ectopic pregnancy. J Reprod Immunol. 2012;93:102–108. 18. Stappers MH, Janssen NA, Oosting M, et al. A role for TLR1, TLR2 and NOD2 in cytokine induction by Bacteroides fragilis. Cytokine. 2012;60:861–869. 19. Lukowski ZL, Min J, Beattie AR, et al. Prevention of ocular scarring after glaucoma filtering surgery using the monoclonal antibody LT1009 (Sonepcizumab) in a rabbit model. J Glaucoma. 2013;22:145–151. 20. Coleman AL. Advances in glaucoma treatment and management: surgery. Invest Ophthalmol Vis Sci. 2012;53: 2491–2494. 21. Yuki K, Kimura I, Shiba D, et al. Elevated serum immunoglobulin G titers against Chlamydia pneumoniae in primary open-angle glaucoma patients without systemic disease. J Glaucoma. 2010;19:535–539. 22. Malamos P, Georgalas I, Rallis K, et al. Evaluation of singledose azithromycin versus standard azithromycin/doxycycline treatment and clinical assessment of regression course in patients with adult inclusion conjunctivitis. Curr Eye Res. 2013;38:1198–1206.
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23. Schaumann R, Funke M, Janssen E, et al. In vitro activities of clindamycin, imipenem, metronidazole, and piperacillin-tazobactam against susceptible and resistant isolates of Bacteroides fragilis evaluated by kill kinetics. Antimicrob Agents Chemother. 2012;56:3413–3416. 24. Inoue T, Kawaji T, Inatani M, et al. Simultaneous increases in multiple proinflammatory cytokines in the aqueous humor in pseudophakic glaucomatous eyes. J Cataract Refract Surg. 2012;38:1389–1397. 25. Chua J, Vania M, Cheung CM, et al. Expression profile of inflammatory cytokines in aqueous from glaucomatous eyes. Mol Vis. 2012;18:431–438. 26. Izzotti A, Sacca` SC, Bagnis A, et al. Glaucoma and Helicobacter pylori infection: correlations and controversies. Br J Ophthalmol. 2009;93:1420–1427. 27. Vohra R, Tsai JC, Kolko M. The role of inflammation in the pathogenesis of glaucoma. Surv Ophthalmol. 2013;58:311–320. 28. Volkov VV. On differences in the pathogenesis, clinical course, treatment, and prevention of glaucoma and ischemic optic neuropathy. Vestn Oftalmol. 2010;126:3–14. 29. Schmier JK, Covert DW. Characteristics of respondents with glaucoma and dry eye in a national panel survey. Clin Ophthalmol. 2009;3:645–650.
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