Study design and study population
All patients aged 30–70 years participating in an in-patient rehabilitation program after acute coronary syndrome (International Classification of Disease, 9th Rev. pos. 410–414) or after coronary artery revascularisation between January 1999 and May 2000 in one of two participating rehabilitation clinics in Germany (Schwabenland-Klinik, Isny and Klinik am Südpark, Bad Nauheim) were enrolled in the study. Germany has a very comprehensive in-patient rehabilitation program and patients who were hospitalized due to acute coronary syndrome (i.e. unstable angina and myocardial infarction) or coronary artery revascularisation (i.e. coronary artery bypass grafting, coronary angioplasty, stent implantation) have the possibility to undergo an in-patient rehabilitation program (rehab) after discharge from the acute care hospital. The aims of this 3-weeks program are the reduction of cardiovascular disease risk factors, improvement of health related quality of life, and the preservation of the ability to work (latter only if a subject was working at onset of disease otherwise prevention of nursing care). This in-patient rehabilitation program begins approximately three weeks after the acute coronary syndrome or the revascularisation procedure but this time interval may be longer in some cases. In the current study only patients who were admitted within 3 months after the acute coronary syndrome or revascularisation procedure were included.
Follow-up was conducted one and three years after discharge from in-patient rehabilitation. All subjects gave written consent to the study. The study was approved by the Ethics Boards of the Universities of Ulm and Heidelberg and of the medical associations of the states of Baden-Wuerttemberg and Hessen.
At the beginning of the in-patient rehab all subjects filled out a standardized questionnaire containing sociodemographic information, smoking status and medical history, including a history of a physician's diagnosis of diabetes mellitus. In addition, information was taken from the patients' hospital charts, and a blood sample was drawn after overnight fast shortly before discharge.
Active follow-up was conducted one and three years after discharge from the rehab clinic. At both times information was obtained from patients via a mailed standardized questionnaire. A standardized questionnaire regarding cardiovascular disease events and treatment since discharge from the in-patient rehabilitation clinic was also obtained from the patient's primary care physician. If a subject died during follow-up, the death certificate was obtained from local Public Health authorities and the main cause of death was coded according to the International Classification of Diseases (9th Revision).
Secondary cardiovascular disease (CVD) events were defined as CVD being main cause of death, or a physician's diagnosis of a non-fatal myocardial infarction or ischemic cerebrovascular event. For the analyses in this study, only subjects with sero-status of Chlamydia pneumoniae (CP), chlamydial heat shock protein (Ch-hsp) 60 and human heat shock protein (h-hsp) 60 and physician's questionnaire at follow up were included. Among 1206 patients with coronary heart disease recruited at baseline, follow-up information by physician's questionnaire or by death certificate could be obtained for 1052 patients (87%), of whom infection sero-status could be determined for 1030 (98%) patients for Chlamydia pneumoniae. H-hsp 60 was available in 983 (93%) patients.
Blood samples drawn before hospital discharge from the rehab clinic were prepared and serum for serology was stored at -80°C. Serum samples were analysed for Chlamydia pneumoniae by using specific enzyme immunoassay for the qualitative and semiquantitative detection of specific IgG or IgA antibodies (Chlamdia pneumoniae-IgG-sELISA medac and Chlamdia pneumoniae-IgA-sELISA medac, medac diagnostika, Hamburg, Germany).
The sero-positivity for the semiquantitative analysis was defined as cut-off index (sample optical density/cut-off) and evaluated as titer. Cut-off index ≤ 1.1 was defined as negative with titer < 1:50, cut-off index >1.1 and ≤ 1.8 was defined as positive with titer 1:50, cut-off index >1.8 and ≤ 3.6 was defined as positive with titer 1:100, cut-off index >3.6 and ≤ 7.2 was defined as positive with titer 1:200. Serum samples were also analysed by using recombinant enzyme immunoassays for chlamydial heat shock protein 60 (cHSP60-IgG-ELISA medac, medac diagnostika, Hamburg, Germany) and human heat shock protein 60 immunoglobulin G antibodies (hHSP60-IgG-ELISA medac, medac diagnostika, Hamburg, Germany). Serum samples were also analysed by using recombinant enzyme immunoassays for chlamydial heat shock protein 60 (cHSP60-IgG-ELISA medac, medac diagnostika, Hamburg, Germany) and human heat shock protein 60 immunoglobulin G antibodies (hHSP60-IgG-ELISA medac, medac diagnostika, Hamburg, Germany). Blood lipids were measured by routine methods and CRP was measured using a high sensitivity CRP (hsCRP) method (N latex CRP mono, Dade Behring, Marburg, Germany). All laboratory measures were done in blinded fashion.
We first described the study population according to basic socioeconomic and medical characteristics. We then calculated the prevalence of sero-positivity to CP, Ch-hsp60 and h-hsp60 at baseline in the whole study population and in various age groups stratified for gender. We quantified possible associations with age by a chi-square statistic.
Furthermore, we described the incidence of secondary cardiovascular disease (CVD) events (cardiovascular death, myocardial infarction and stroke) and their combination during follow up. The occurrence of fatal and non-fatal CVD events during follow up according to sero-prevalence of CP, Ch-hsp60 and h-hsp60 at baseline was analysed by the life table method.
Finally, we used the Cox proportional hazards model to assess a possible association of sero-positivity to CP, Ch-hsp60 and h-hsp60 and diabetes (alone and in combination) at baseline with fatal and non-fatal CVD events during follow up while adjusting for the following covariates: age (years), gender (male, female), HDL-cholesterol (>40, ≤ 40 mg/dl), history of smoking (never, ever), alcohol consumption within last 12 months (none, any), school education (≤ 9 years, >9 years), marital status (married, other), history of myocardial infarction (never, ever), study centre (Bad Nauheim/Isny). Associations were quantified by hazard ratios (HR) and their 95% confidence interval (CI). In addition, a possible interaction between CP, Ch-hsp60 and h-hsp60 sero-status and diabetes was assessed by introducing a product term of each of the infection markers and history of diabetes in the adjusted model. In addition, age and gender adjusted geometric mean values of CRP (which is considered as a feature of atherosclerosis) were calculated and compared according to CP, Ch-hsp60 and h-hsp60 sero-status and history of diabetes by means of General Linear Regression methods. All analyses were carried out using the SAS software package, version 8.2.