Chlamydia pneumoniae is a common cause of respiratory infections worldwide [6, 7]. Epidemiologic studies have indicated an association between atherosclerosis and C. pneumoniae infection, and this pathogen has been found in atherosclerotic lesions [8-12]. In addition, all Chlamydia species have been observed to cause heart infections [13, 14]. Chlamydia pneumoniae is frequently found in stenotic aortic valves and in the early lesions associated with this disease, but it is rarely found in normal aortic valves of age-matched controls [15]. Chlamydia pneumoniae may merely be an innocent bystander occupying damaged tissues; however, because this organism is known to cause persistent infections, it may trigger and maintain the chronic inflammation found in aortic stenosis.

We explored the association between C. pneumoniae infection and aortic stenosis. We tested the hypothesis that if persistent C. pneumoniae infection plays an active role in the development of aortic stenosis, the organism can be detected in the healthy aortic valves of young persons.

Methods

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Patients

Aortic valves were obtained from 46 consecutive cadavers undergoing autopsy at the Departments of Forensic Medicine and Pathology at Oulu University Hospital and University of Oulu, Oulu, Finland. The ascending aorta was incised longitudinally, the aortic valve was exposed, and the leaflet movements were evaluated. The valve and its annulus were excised. In accordance with previous studies [4, 15], macroscopic disease was classified as absent (translucent, flexible leaflets with no opaque area), as a mild or early lesion (opaque leaflets with focal areas of thickening and increased stiffness but no obstruction), or as severe (definite areas of calcification and thickening with substantial obstruction). The macroscopic analyses were performed by two of the authors, who were blinded to other aspects of the investigation. The ethical committee of Oulu University approved the study.

Immunohistochemistry

The noncoronary leaflets of the valves were immersed in 10% buffered formalin for at least 24 hours and were embedded in paraffin for immunohistochemical and histologic studies, which were performed in a blinded manner. For immunohistochemical studies, 4-µm to 5-µm slices were cut, deparaffinized, rehydrated, and digested with pepsin. No other antigen retrieval methods were used. Slices were then treated with H (₂) O₂ to remove peroxidase activity and with normal serum to remove nonspecific binding of the antibody. The specimens were incubated with C. pneumoniae monoclonal antibody RR402 (Washington Research Foundation, Seattle, Washington) in 1:200 dilution for 1 hour at room temperature and were washed carefully. Binded antibody was detected by using the avidin-biotin-peroxidase method of Hsu and colleagues [16] with a Vestastain ABC kit (Vector Laboratories, Burlinggame, California). Diaminobenzidine was used as substrate. HL cells infected with C. pneumoniae were used as positive controls, and normal aorta and myometrium were used as negative controls. Only granular intracellular staining was accepted as positive.

Statistical Analysis

To study the possible persistence of C. pneumoniae in aortic valves, the data were stratified by age (persons 20 to 40 years of age [n = 15], persons 41 to 60 years of age [n = 16], and persons older than 60 years of age [n = 15]). The two-sided Fisher exact test was used to determine whether probability of early lesion was related to the presence of C. pneumoniae in the aortic valve. A P value less than 0.05 was considered significant. We calculated 95% CIs for differences in proportions by using the exact method in StatXact3 for Windows (Cytel Software Corp., Cambridge, Massachusetts).

Results

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The mean age at death was 50 years (range, 22 to 83 years). Thirty persons were men and 16 were women. All of the aortic valves were tricuspid, and none was stenotic. Thirty-four of the 46 valves were macroscopically normal. Early lesions were detected in 12 valves (0 of 15 valves from persons 20 to 40 years of age, 4 of 16 valves from persons 41 to 60 years of age, and 8 of 15 valves from persons older than 60 years of age; P = 0.004). In one case, mild aortic insufficiency was recorded at autopsy. Histologic examination of the 12 valves with early lesions showed degeneration, calcification, and variable inflammatory cell infiltrates. Some of the macroscopically normal valves had degenerative changes on microscopic analysis. Positivity for C. pneumoniae was seen in stromal spindle and mononuclear cells and was spread diffusely in the valve tissue. In normal valves, scattered macrophages but no T cells were detected. Valves with early lesion were characterized by the presence of an inflammatory infiltrate composed of macrophages and occasional T cells.

A positive result on staining was not clearly associated with inflammatory reactions, although both inflammation and positivity for C. pneumoniae increased with age and were more frequently found in valves with early lesions. Macroscopically and microscopically normal valves without inflammation tested positive for C. pneumoniae. A typical positive immunostaining is shown in the (Figure 1). Twenty-five valves (54%) had positive results on staining (15 of 34 macroscopically normal aortic valves [44%] and 10 of 12 valves with early lesions [83%]; P = 0.02). The relations of age and result of staining for C. pneumoniae to the presence of early lesions are shown in the (Table 1). Nine of 15 valves (60%) from persons 20 to 40 years of age, 8 of 16 valves (50%) from persons 41 to 60 years of age, and 8 of 15 valves (53%) from persons older than 60 years of age had a positive result on staining. No person younger than 40 years of age had early lesions, and the chance of early lesions in persons 41 to 60 years of age tended to be greater if C. pneumoniae was present in the aortic valve (3 of 4 [75%] aortic valves with C. pneumoniae infection compared with 5 of 12 [42%] aortic valves without C. pneumoniae infection; P > 0.2). Persons older than 60 years of age had a significantly greater chance of early lesion if they had C. pneumoniae infection (7 of 8 [87%] aortic valves with C. pneumoniae infection compared with 1 of 7 [14%] valves without C. pneumoniae infection; P = 0.01). Among macroscopically normal valves, 9 of 15 (60%) from persons 20 to 40 years of age, 5 of 12 (42%) from persons 41 to 60 years of age, and 1 of 7 (14%) from persons older than 60 years of age had positive results on staining.

View larger version (122K): [in this window] [in a new window]   Figure 1. A positive result on immunohistochemical staining with Chlamydia pneumoniae species specific antibody in mononuclear cells (arrows) of a macroscopically normal aortic valve. Original magnification, x200.

Discussion

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Our results show that C. pneumoniae is frequently found in the aortic valves of adults of different ages and that the presence of this pathogen is associated with aortic valve disease in elderly persons. This finding suggests the persistence of C. pneumoniae in normal aortic valve tissue.

The persistence of Chlamydia species is a well-known phenomenon [17-19]. It has long been known that C. trachomatis can be detected by immunofluorescence in the eyes of many persons who do not have any signs of active disease [17-20], and the persistence of C. pneumoniae in throat swab specimens has been shown [20]. Our findings show that C. pneumoniae can infect the normal aortic valve and that infection may persist. The 50% rate of positivity for C. pneumoniae in aortic valves is in line with the overall prevalence of the antibody. Chlamydia pneumoniae infection is very common in children, and the prevalence of antibodies indicating past infection is about 50% in young adults. In older age groups, the prevalence of antibodies increases to as much as 75% [2]. The antibody response is limited to 3 to 5 years; this suggests that most people become infected several times during their lives.

Chlamydia pneumoniae has been found in atherosclerotic lesions and stenotic aortic valves, but it has not been found in normal vessel walls or in the occluded coronary arteries of patients in whom transplants are chronically rejected [5-7]. This has raised the question of whether C. pneumoniae infects only atherosclerotic vessels or is an innocent bystander. Most of the C. pneumoniae-positive valves in our study were macroscopically normal, but C. pneumoniae was rarely found in normal valves in elderly persons (as observed in our previous study [15]). The finding that C. pneumoniae transfers from normal valves to valves with early lesions over the years suggests the pathologic potential of the organism. The difference between the incidence of C. pneumoniae in normal valves and valves with early lesions supports a possible causal relation.

What could be the role of latent chlamydial infection in the pathogenesis of degenerative aortic stenosis? Of interest, a chlamydial antigen was reported to be present in aortic stenosis in 1974 [14]. In those patients, the diagnosis was based on results of an indirect immunofluorescence test that used polyclonal anti-C. psittaci antibodies, which probably contained genus-specific antibodies that reacted not only with C. psittaci but also with C. pneumoniae and C. trachomatis [13]. There is little evidence of the clinical significance of persistent chlamydial infections. The host response to the infection seems to be a major contributor to the development of the disease. Repeated or persistent infections as such provide an opportunity for chronic stimulation of the host with chlamydial antigens and are associated with a poor outcome. For example, primary ocular infection with C. trachomatis in humans and monkeys resolves with little or no residual tissue damage, but recurrent infections produce intensive inflammatory reactions that cause scarring trachoma. Trauma or immunologic changes may also reactivate quiescent chlamydial infection [17-20]. By analogy to trachoma, it is tempting to speculate that C. pneumoniae may persist in the aortic valves of some immunologically vulnerable persons after a primary infection at an early age. The trauma that activates this infection could be caused by mechanical stress, or several C. pneumoniae reinfections could activate immunologic responses. In addition, bacteria or bacterial antigens can feed the inflammation, stimulate the production of inflammatory cytokines, and provoke and contribute to endothelial injury [17].

Drs. T. Juvonen and Kairaluoma: Department of Surgery, Oulu University Hospital, FIN-90220 Oulu, Finland.

Dr. Laurila: Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0682.

Dr. Kuusisto: Department of Internal Medicine, Kuopio University Hospital, FIN-70211 Kuopio, Finland.

Dr. Alarakkola: Department of Pathology, Tampere University Hospital, FIN-33521 Tampere, Finland.

Dr. Sarkioja: Department of Forensic Medicine, University of Oulu, FIN-90220 Oulu, Finland.

Dr. Bodian: Department of Biomathematics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029.

Dr. Saikku: National Public Health Institute, FIN-90220 Oulu, Finland.