Exposure to Chlamydia pneumoniae Infection and Progression of Age-related Macular Degeneration
Luba Robman1,
Olaimatu Mahdi2,
Catherine McCarty1,3,
Peter Dimitrov1,
Gabriella Tikellis1,
John McNeil4,
Gerald Byrne2,
Hugh Taylor1 and
Robyn Guymer1
1 Centre for Eye Research Australia, University of Melbourne, Melbourne, Australia
2 Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN
3 Marshfield Clinic Research Foundation, Marshfield, WI
4 Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia
Correspondence to Dr. L. Robman, Centre for Eye Research Australia, University of Melbourne, 32 Gisborne Street, East Melbourne 3002, Australia (e-mail: liubov{at}unimelb.edu.au).
Received for publication September 29, 2004.
Accepted for publication January 19, 2005.
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ABSTRACT
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Recent studies have found an association between exposure to Chlamydia pneumoniae infection and risk of age-related macular degeneration (AMD). To assess a potential risk of AMD progression posed by exposure to C. pneumoniae, the authors reexamined Australian residents in 20012002 who were aged 5189 years with early AMD at baseline (19921995). Examination included macular photography and an enzyme-linked immunosorbent assay to determine antibody titers to the elementary bodies from C. pneumoniae AR39. AMD progression was assessed quantitatively, using both coarse and fine progression steps following an international classification for AMD grading, and also qualitatively, by side-by-side comparison of baseline and follow-up macular photographs. Serologic data were available for 246 of 254 (97%) subjects. AMD progression was associated with a higher antibody titer. After adjustment for age, smoking, family history of AMD, history of cardiovascular diseases, and source study, the subjects in the upper tertiles of antibody titers were 2.1 (95% confidence interval: 0.92, 4.69), 2.6 (95% confidence interval: 1.24, 5.41), and 3.0 (95% confidence interval: 1.46, 6.37) times more at risk of progression than those in the lowest tertile, using three definitions of progression, respectively. The fact that seroreactivity to C. pneumoniae was independently associated with the risk of AMD progression suggests that C. pneumoniae infection may be an additional risk factor for AMD progression.
Chlamydophila pneumoniae; disease progression; infection; macular degeneration
Abbreviations:
AMD, age-related macular degeneration; VECAT, Vitamin E, Cataract, and Age-related Maculopathy Trial
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INTRODUCTION
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Age-related macular degeneration (AMD) is the leading cause of unavoidable visual loss in people over 60 years of age in Australia and across industrialized nations (1
3
). The disease affects central vision and is responsible for one third of untreatable vision loss. Early AMD is characterized by lipid deposits (drusen) and pigmentary abnormalities at the macula (central retina). In late AMD, the macula is damaged either through atrophy or by growth of new blood vessels. AMD is not preventable and for most patients it is untreatable. The prevalence of AMD increases dramatically with age (4
8
), resulting in enormous personal costs as well as being a significant burden on health resources. Although age and family history are well-established risk factors, to date, smoking is the only modifiable risk factor associated with AMD (9
). Thus, much of the attributable risk remains unexplained.
We wished to explore the possible epidemiologic association between progression of AMD and exposure to the Chlamydia pneumoniae infection. AMD has been associated with cardiovascular disease and atherosclerosis and their risk factors (10
). Cardiovascular disease and atherosclerosis have been considered as having an inflammatory component, and a number of studies suggested an association of these diseases with C. pneumoniae infection (11
18
). As a result of these findings, an association was sought and found between AMD and exposure to the C. pneumoniae infection (19
, 20
). Chlamydial pneumonia DNA has recently been identified in neovascular tissue surgically removed from eyes with AMD (21
). C. pneumoniae has also been associated with other ophthalmic diseases, such as acute anterior uveitis (22
) and nonarteritic ischemic optic neuropathy (23
). We investigated the possibility of an association between exposure to C. pneumoniae infection and progression of AMD.
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MATERIALS AND METHODS
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Study design and subject selection
The Cardiovascular Health and Age-related Maculopathy Study (24
) enrolled in 2001 and 2002 participants with early AMD identified at baseline examination between 1992 and 1995. Participants were chosen from two previous studies conducted in Melbourne, Australia: 1) the population-based Melbourne Visual Impairment Project (25
), where baseline examinations were conducted in 19921994, and 2) the randomized controlled trial entitled the Vitamin E, Cataract, and Age-related Maculopathy Trial (VECAT), where baseline examinations were conducted in 1995 (26
). The VECAT found no effect of vitamin E on the course of AMD (27
), and therefore AMD cases from this cohort were incorporated into the Cardiovascular Health and Age-related Maculopathy Study. These two studies used the same study methodology to document retinal features and determine risk factors for disease. Subjects were classified as eligible if they had features of early AMD (intermediate drusen, soft drusen, and retinal pigment epithelium abnormalities) in at least one eye.
Details of the recruitment strategies and enrollment results have been recently described (24
). The study protocol was approved by the Royal Victorian Eye and Ear Hospital Human Research and Ethics Committee (project 99/372H). Written informed consent was obtained from each participant. All subjects were required to attend the study center and to complete the full examination as required by the study protocol.
Macular photography and grading
Stereo photography of the macula was performed on Kodachrome 64 ASA 35-mm slide film (Eastman Kodak Company, Rochester, New York), following pupil dilatation with tropicamide (0.5 percent) and phenylephrine hydrochloride (10 percent). Grading of macular characteristics was carried out according to an international classification and grading system for AMD by the International ARM [Age-related Maculopathy] Epidemiological Study Group (28
). Each slide was graded for the absence or presence, size, location, number, centrality, and area covered by the following features: hard, intermediate, distinct, and indistinct drusen; retinal pigment epithelium changes; geographic atrophy; and neovascular age-related maculopathy (29
, 30
). All the baseline photos were regraded to avoid time-related shift in grading. The macular graders were masked to the serologic data.
Early AMD status at baseline and follow-up was stratified into a six-level severity scale, described elsewhere (27
): level 1, no drusen/hard drusen only; level 2, intermediate drusen or hyperpigmentation without hypopigmentation; level 3, soft distinct or indistinct drusen or pigmentary abnormalities (hyper- and hypopigmentation or hypopigmentation alone); level 4, soft distinct or indistinct drusen and pigmentary abnormalities (hyper- or hypopigmentation); level 5, geographic atrophy; and level 6, neovascular AMD. Levels 24 at baseline were eligible to assess progression.
With regard to progression of AMD, to determine an association, the data were analyzed using three different definitions of AMD progression, two quantitative and one qualitative, to ensure the robustness of the finding. Initially, AMD progression was defined broadly as an increase from level 2, 3, or 4 at baseline by one or more levels in the worse affected eye (definition 1).
To allow a finer definition of progression, those cases where there was an increase from level 2, 3, or 4 at baseline by one or more levels in either eye and also the cases with an increase of two or more steps in the specific grades (28
) used to assess size, total number, area occupied by a lesion, and spread to a more central location within a level were defined as progressed (definition 2).
Finally, side-by-side grading was used to allow a qualitative definition of progression, which was defined as a worsening of the AMD status assessed through a masked side-by-side comparison of the baseline and follow-up macular photographs (definition 3).
Serologic tests
Serum was collected at the follow-up examination and stored in plastic cryotubes at 80°C (Sarstedt AG & Co., Nümbrecht, Germany) at the study center. Serum samples (0.5 ml) were forwarded on ice to the Department of Molecular Sciences, University of Tennessee Health Sciences Center (Memphis, Tennessee). Enzyme-linked immunosorbent assays were performed using elementary bodies from C. pneumoniae AR39, and titers were expressed as absorbance units. Details of enzyme-linked immunosorbent assay protocol have been previously described (19
).
For each serum sample, the absorbance value of a phosphate-buffered, saline-coated well that had no antigen (antigen blank) was subtracted from the values for all the test wells for that antigen. Triplicate-blanked test absorbance values were averaged and reported for each patient. Laboratory personnel were masked to clinical and demographic information on the patients.
Statistical analysis
Univariate logistic regression analysis was used to determine association of the potential risk factors with the progression of AMD. The averages of the logged antibody titers in the two groups were compared to detect a trend for an association. Multivariate logistic regression analysis was used to determine the strength of the association between the antibody titers to C. pneumoniae and AMD progression. Statistical analyses were performed using the SPSS statistical software package (SPSS, Inc., Chicago, Illinois). A 5 percent level of significance (two sided) was used.
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RESULTS
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Demographics
The 254 subjects enrolled for assessment of AMD progression were aged 5189 years (mean age, 74 (standard deviation, 7) years); 135 (53 percent) participants were female, 117 (46 percent) were former or current smokers, and 12 (4.7 percent) had a family history of AMD (table 1). There were more participants with family history of AMD among progressors, the age of those who progressed was about 3 years older, and the percentage of smokers among them was higher.
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TABLE 1. Univariate logistic regression analysis of associations between age-related macular degeneration progression and potentially confounding risk factors (n = 254), Melbourne, Australia (20002002), and Memphis, Tennessee (2003)
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AMD grading
The association between AMD progression and seroreactivity to C. pneumoniae infection was analyzed using each of the three definitions described for assessing progression. There were 61 cases of progression defined as an advance in at least one level of AMD severity. Of them, 15 (25 percent) cases progressed from early AMD to the late stages. Using the fine grading, which included cases with an advance in at least one level of AMD severity in either eye and also cases that progressed within a level, there were an additional 21 (8 percent) cases that progressed, bringing the total to 82 (33 percent). Side-by-side assessment identified progression in 83 (33 percent) cases. There were nine (3.5 percent) cases of disparity in assessment of progression between the last two definitions of progression. The rate of progression in the group of participants enrolled from the VECAT was higher than in those enrolled from the Melbourne Visual Impairment Project (30 percent vs. 20 percent, 38 percent vs. 28 percent, and 36 percent vs. 30 percent, for the three definitions of progression, respectively). Although the difference was not statistically significant, this variable was included in the final model, to account for its possible confounding effect.
Serology
Serologic data were available for 246 of 254 (97 percent) subjects. After logarithm transformation, the antibody titers were compared with AMD progression. This comparison showed an increase in antibody titers with AMD progression, using all three definitions of progression. There was an insignificant increase in the antibody titers in progressed cases using the definition of an increase of at least one level in AMD severity (ptrend
0.23). When progression was assessed by adding in the cases who progressed within a level in AMD severity, there was a larger number of cases with low antibody titers in the group that did not progress (figure 1), and the increase in the antibody titers in those that did progress was statistically significant (p
0.014). A similar increase in the antibody titers in those AMD cases that progressed was seen when using side-by-side comparison (p
0.003).

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FIGURE 1. Distributions of the titers of antibodies to Chlamydia pneumoniae elementary bodies in the groups of progressed and nonprogressed cases of age-related macular degeneration (AMD), Melbourne, Australia (20002002), and Memphis, Tennessee (2003). Fine grading, definition 2 for AMD progression: n = 246.
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Logistic regression analysis was performed to quantify the strength of the association between seropositivity to C. pneumoniae and progression of AMD. An increase in antibody titers to C. pneumoniae whole elementary bodies was associated with AMD progression after controlling for age, smoking, family history of AMD, history of cardiovascular diseases, and the source study. Subjects in the two upper tertiles of antibody titer were at a significantly greater risk of AMD progression than those in the lowest tertile. The twofold increased risk of progression for the subjects in the middle tertile of the antibody titers was consistent for all three definitions of AMD progression (table 2). In the upper tertile of antibody titers, the risk of progression had a further increase, reaching an odds ratio of 2.6 (95 percent confidence interval: 1.24, 5.41) when using the fine inter- and intralevel definitions of progression and an odds ratio of 3.0 (95 percent confidence interval: 1.46, 6.37) when using side-by-side grading for progression.
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TABLE 2. Multivariate logistic regression analysis of the association of age-related macular degeneration progression with seropositivity to Chlamydia pneumoniae elementary bodies (n = 246), Melbourne, Australia (20002002), and Memphis, Tennessee (2003)
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DISCUSSION
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C. pneumoniae is an obligate intracellular pathogen responsible for 620 percent of cases of community-acquired pneumonia (31
33
). Most infections are mild or asymptomatic, and the 4090 percent prevalence of positive serology indicates a high level of community exposure (32
, 34
36
). The organism is thought to be disseminated through the bloodstream and has a particular attraction for vascular tissue, where it may establish a chronic infection (37
). There is evidence that chronic inflammation may be involved in the development of AMD (19
, 38
, 39
).
In this study, we have shown that the rate of progression of AMD over a 7-year period was increased in those with higher titers of antibodies to C. pneumoniae. Depending on the definition of progression, individuals whose antibody titers were in the upper tertile of the distribution were from two to three times more likely to progress than those in the lowest tertile. This result was present after controlling for 1) differences in age and smoking status, the two major factors known to be independently associated with the prevalence and progression of AMD; 2) family history of AMD, since this appeared to be associated with progression and may reflect heridability of the disease; and 3) history of heart attack, stroke, and/or high blood pressure, since cardiovascular disease may possibly be associated with both the outcome and covariate of interest. These results raise the question of whether C. pneumoniae may play a causal role in the development of AMD.
The findings of this study are in keeping with those of cross-sectional studies that have also demonstrated an association between C. pneumoniae infection and AMD. In 2004, Kalayoglu et al. (21
) reported finding C. pneumoniae in five of nine specimens of AMD but in none of nine normal eyes. In two cross-sectional studies, Kalayoglu et al. (19
) and Ishida et al. (20
) found the prevalence of antibodies to C. pneumoniae to be significantly higher in subjects with AMD compared with age-matched controls. Our study adds to this work by showing that the presence of these antibodies is linked to more rapid progression of AMD, thus increasing the likelihood of a causal role.
C. pneumoniae has also been linked to atherosclerosis. An increased frequency of positive C. pneumoniae serology has been noted in several epidemiologic studies comparing patients with and without large-vessel disease (40
). The C. pneumoniae organisms were detected in 67 percent of vessels that showed only early atherosclerotic lesions (fatty streaks), whereas 71 percent of atheromatous arteries taken at autopsy from White South African subjects were C. pneumoniae positive compared with 9 percent of nonatheromatous arteries (17
). Atherosclerosis shares risk factors in common with AMD and may also have a similar pathophysiologic basis.
In an attempt to determine whether C. pneumoniae plays a significant causal role in the development of atheroma, several large trials of long-term antibiotic therapy designed to eradicate C. pneumoniae have been undertaken (41
). None of these trials has shown any significant reduction of subsequent vascular events, even in those with the highest titers of anti-C. pneumoniae antibodies. This suggests that C. pneumoniae infection is not a common ongoing stimulus to atheroma progression but leaves open the possibility that the organism may trigger the onset of an inflammatory response, at least in some patients. Alternatively, C. pneumoniae may simply be a commensal organism that resides in damaged blood vessels without playing any pathologic role.
The role of C. pneumoniae in AMD will also require further study to elucidate its relation to this condition. This should involve confirmation of the present results in other populations. If the relation is confirmed, long-term clinical trials of antibiotic therapy similar to those undertaken in cardiovascular disease will be necessary to confirm or refute a pathogenic role for these organisms.
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ACKNOWLEDGMENTS
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The Cardiovascular Health and Age-related Maculopathy Study was supported by National Health and Medical Research Council grant 128201. Serologic tests were conducted in the United States and supported by Public Health Service grant AI 42790 from the National Institutes of Health. Additional support was provided by Perpetual Trustees Australia, Ltd. (Ramaciotti Foundation), ANZ Executors & Trustee Company, Ltd. (The Hugh D. T. Williamson Foundation under the Medical Research and Technology in Victoria Program), the Royal Victorian Institute for the Blind, the Royal Victorian Eye & Ear Hospital Research Committee, the Lions Club of Victoria, and the Australian Institute of Health and Welfare.
The authors declare no conflict of interest.
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