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Antibodies to Chlamydia pneumoniae and clinical course in patients with unstable angina pectoris
Atherosclerosis 153 (2000) 499 – 504 www.elsevier.com/locate/atherosclerosis
Antibodies to Chlamydia pneumoniae and clinical course in patients with unstable angina pectoris Moniek P.M. de Maat a,*, Jacobus M. Ossewaarde b, Peter W.H.M. Verheggen c, Cornelis Kluft a, Volkert Manger Cats c, Frits Haverkate a a
Di6ision of Vascular and Connecti6e Tissue Research, Gaubius Laboratory TNO-PG, PO Box 2215, 2301 CE, Leiden, The Netherlands b Research Laboratory for Infectious Diseases, National Institute of Public Health and the En6ironment, Biltho6en, The Netherlands c Department of Cardiology, Leiden Uni6ersity Medical Centre, Leiden, The Netherlands Received 13 September 1999; received in revised form 19 January 2000; accepted 11 February 2000
Abstract Inflammation is one of the most important mechanisms that contribute to coronary artery disease (CAD). One of the micro-organisms that is mentioned as a source of the inflammation is Chlamydia pneumoniae. In this study, we investigated the relationship between titres of IgG and IgA antibodies to C. pneumoniae and the clinical course, during hospitalisation and during an 18-month follow-up, in 211 patients admitted to hospital with unstable angina pectoris. Slightly more patients who were refractory during their hospitalisation were positive for C. pneumoniae antibodies than patients who could be stabilised by drug treatment (53 vs. 43%, for IgG and 16 vs. 11% for IgA, respectively)(n.s.). In logistic regression analysis no significant predictive values were observed for the relationship between antibody titres and clinical course. The antibody titres to C. pneumoniae were lower in the unstable angina patients who had plasma levels of interleukin-10 (IL-10) above 5 pg/ml than in the patients with levels below 5 pg/ml, and higher in smokers than in non-smokers. No associations were observed between antibody titres to C. pneumoniae and C-reactive protein (CRP), interleukin-6 (IL-6), age, total cholesterol levels, fibrin degradation products (FDP), plasminogen activator inhibitor-1 (PAI-1) and erythrocyte sedimentation rate (ESR). In conclusion, there was no significant association between antibody titres to C. pneumoniae and risk of events during hospitalisation and the 18-month follow-up period in patients admitted for unstable angina pectoris. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Unstable angina; Chlamydia pneumoniae; Inflammation; C-reactive protein; Interleukin-10
1. Introduction Inflammation is one of the most important mechanisms that contribute to the severity and the progression of coronary artery disease (CAD) [1]. Furthermore, the blood levels of the inflammatory marker fibrinogen [2] and C-reactive protein (CRP) [3,4] are associated with the risk of cardiovascular events. The association between blood levels of inflammatory markers and risk may reflect the inflammatory state of the vascular wall, but it is also possible that they are a reflection of a systemic inflammation. One cause of systemic inflammation may be chronic infec* Corresponding author. Tel.: +31-71-5181502; fax: + 31-715181904. E-mail address: [email protected] (M.P.M. de Maat).
tion. Indeed, some types of infection such as Chlamydia pneumoniae have been associated with cardiovascular risk [5]. Since 1989 C. pneumoniae has been established as a third species of the genus Chlamydia. On the basis of clinical research in the past 15 years, it is now estimated, that C. pneumoniae causes an average of 10% of cases of community-acquired pneumonia and 5% of bronchitis [6]. In the Netherlands, the prevalence of antibodies to C. pneumoniae in adults is 60–80% [7,8]. In 1988, Saikku and co-workers associated C. pneumoniae with CAD [9]. This association has been confirmed in several cross-sectional and prospective studies [8,10,11]. Mechanisms that have been suggested to underlie the association between C. pneumoniae and CAD are (a) that Chlamydia lipopolysaccharide (LPS) contributes to the formation of foam cells and (b) that
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M.P.M. de Maat et al. / Atherosclerosis 153 (2000) 499–504
Chlamydia heatshock protein 60 activates macrophages [12 – 14]. In this study, we investigated the relationship between antibody titres to C. pneumoniae and the clinical course, in-hospital and during an 18-month follow-up, in 211 patients admitted to the hospital with unstable angina pectoris.
2. Patients and methods
2.1. Study population Acute phase reaction and ischaemic syndromes (APRAIS) is a prospective study on 211 patients with unstable angina pectoris, consecutively admitted to the Leiden University Medical Center. Details of the study population and recruitment have been published elsewhere [15]. Briefly, patients were included when they experienced typical angina at rest within 24 h before admission in combination with either, (1) myocardial ischaemia as reflected in ST-segment depression; or (2) electrocardiographic evidence of CAD based on prior Q-wave myocardial infarction; or (3) previous coronary angiography showing at least 50% lumen diameter narrowing in at least one large epicardial coronary artery. Patients with intercurrent inflammatory or neoplastic disease likely to be associated with acute phase responses were excluded. All patients received standard medical therapy unless contra-indicated and all decisions regarding the care of patients were made without knowledge of the biochemical study variables. The end point of the in-hospital follow-up was the occurrence of refractory unstable angina. Unstable angina was considered to be refractory if, in spite of optimal medical therapy, angina at rest reoccurred and led to the decision to perform urgent coronary angiography in order to assess the chances of successful coronary angioplasty or bypass surgery. The end point of the 18-month follow-up was a cardiac event (acute myocardial infarction, cardiac death, bypass surgery, coronary angioplasty, refractory unstable angina). The Ethics Committee of the Leiden University Medical Center approved the study protocol.
2.2. Assay procedures On admission, prior to the start of medication, venous blood was collected in glass tubes without additives and in sodium citrate (final concentration 0.011 mol/l). After complete clotting of the glass tubes, the blood was centrifuged for 30 min at 2000 × g at room temperature. The tubes with citrate were immediately placed in melting ice and centrifuged 30 min at 2000× g and 4°C. Serum and plasma were collected and stored in aliquots at −70°C until used.
IgG and IgA antibodies to C. pneumoniae have been determined using an enzyme immunoassay with minor modifications as described previously [16]. Briefly, C. pneumoniae strain TW183 was used to infect monolayers of HEp2 cells in six-well microtitre plates. After 3 days’ incubation, the antigen was purified by centrifugation through a layer of 35% sodium diatrizoate. Control antigen was prepared by the same method, but without infection of the monolayers. The antigens were used to coat microtitre plates. The genus-specific LPS antigen was destroyed in half of the microtitre plate by incubation with 1% sodium periodate in phosphatebuffered saline (PBS), pH 7.2. Destruction of the LPS results in a more specific assay [17,18]. A reference serum was included on each microtitre plate. Patient specimens were tested in twofold and the titre was calculated relative to the reference serum. CRP was measured in serum by a nephelometric method, range 0.2–1100 mg/l (N Latex CRP mono, Dade-Behring). Interleukin-6 (IL-6) was determined by a highly sensitive enzyme immunoassay, range 0.16–10 ng/l (Quantikine HS IL-6, R&D Systems, Oxon, UK). Interleukin-10 (IL-10) was determined by an enzyme immunoassay [19]. Fibrinogen was determined according to the method of Von Clauss ([20] 1957) using pooled citrated plasma (n= 25) in which the fibrinogen level was determined by a gravimetric method as reference material [21]. Erythrocyte sedimentation rate (ESR) was measured by the modified Westergren method. Plasminogen activator inhibitor (PAI-1) activity levels were determined using the method described by Verheijen et al. [22]. Fibrin(ogen) degradation products (FDP) concentrations were determined using an ELISA (Fibrinostika TDP, Organon Teknika, Turnhout, Belgium).
3. Statistical analysis Serum antibody titres to C. pneumoniae, PAI-1 and CRP were logarithmically transformed before analysis to normalise the distribution. The incidence of positive antibody titres was determined after dichotomization of the titre in 1:B 3200 and 1:] 3200. Three thousand two hundred was chosen as cut-off to exclude crossreactions with other Chlamydia species. This has also been done in the Zutphen study [8] and there it proved to be correct cut-off the use as prognostic marker. Logistic regression analysis was used to determine the odds ratio of having an event with titres 1:]3200 relative to a titre 1: B 3200, after adjustment for age and gender. The power of study to detect a relative risk of refractiveness (in-hospital) of 2.0 was 0.66 for IgG and 0.40 for IgA, for events during the 18-month
M.P.M. de Maat et al. / Atherosclerosis 153 (2000) 499–504
follow-up the power was 0.66 for IgG and 0.42 for IgA, respectively. Pearson correlation coefficients were calculated to study the association between antibody titres to C. pneumoniae and other inflammatory variables. The relationship between antibody titres to C. pneumoniae and in-hospital and the 18-month follow-up was studied using logistic regression, with C. pneumoniae as a continuous variable or as a dichotomised variable (above or below a titre of 3200). A Student’s t-test on logarithmically transformed data was used to compare the levels in smokers and non-smokers. All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) for Windows, version 8.0 (SPSS Inc.).
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odds ratio of becoming refractory with a high antibody titre to C. pneumoniae on admittance was somewhat, but not significantly, higher than 1 for both IgA and IgG (1.31 (95% CI 0.55, 3.10) and 1.37 (95% CI 0.74, 2.50), respectively) (Table 1). When the relationship between antibodies to C. pneumoniae on admission and the clinical course after 18month follow-up were studied there was no significant difference in IgG antibodies in patients with or without a cardiac event but there were still somewhat more patients with increased IgA antibodies in those patients who experienced an event (Table 2). Also, in logistic regression analysis no significant predictive value of any of the baseline antibody titres to C. pneumoniae for events in the 18-month follow-up period was observed (Table 2).
4. Results
4.1. Relationship between antibody titres to C. pneumoniae and clinical course
4.2. Relationship of C. pneumoniae antibodies with inflammatory markers and other risk factors for cardio6ascular disease
Antibody titres to C. pneumoniae could be determined in 207 patients. Of these, 132 could be treated medically, and the remaining 75 patients required urgent coronary angiography because of persistent, refractory unstable angina. During the 18-month follow-up, a cardiac event occurred in 113 patients. More patients who were refractory during their hospitalisation had antibody titres to C. pneumoniae 1:] 3200 than patients who could be stabilised by drug treatment (Table 1). For IgG antibodies (both for native and for periodate-treated elementary bodies (EBs)) the excess of patients with high antibody titres was about 10%, for IgA antibodies the excess was 4%. However, these differences were not significant. The
The antibodies to native C. pneumoniae EBs on admittance to the hospital with UA were significantly higher in patients with a history of smoking than in non-smokers (geom. mean titres (25–75% range) for IgA antibodies were 821 (362–1637) versus 539 (295– 742), P= 0.006 and for IgG antibodies 4866 (1766– 12386) versus 2981 (1184–5129), P= 0.007. Using the titres to sodium periodate-treated C. pneumoniae gave similar results (geom. mean titre for IgA after periodate treatment 889 (355–2193) versus 602 (316–1158), P= 0.02 and for IgG 4131 (1734–9069) versus 2598 (1221– 4498), P= 0.005). In patients who had plasma levels of IL-10 above 5 pg/ml on admittance to the hospital with unstable
Table 1 Relationship between antibody titres to C. pneumoniae and refractiveness during hospitalisation of patients with unstable anginaa
IgA IgA IgG IgG a
(1:]3200) periodate (1:]3200) (1:]3200) periodate (1:]3200)
Stabilised (n = 132)
Refractory (n =75)
P value
OR (95% CI)
11 15 70 57
9 12 46 40
0.46 0.39 0.31 0.19
1.37 1.31 1.27 1.37
(8%) (11%) (53%) (43%)
(12%) (16%) (61%) (53%)
(0.52, (0.55, (0.70, (0.75,
3.58) 3.10) 2.31) 2.50)
Given are the number of patients with antibody titres 1:\3200 and percentage (%). Odds ratios (OR) are adjusted for age, sex.
Table 2 Relationship between antibody titres to C. pneumoniae and events during 18 months after admission in patients with unstable anginaa
IgG IgG IgA IgA a
(1:]3200) periodate (1:]3200) (1:]3200) periodate (1:]3200)
No events (n=94)
Events (n = 113)
P value
OR (95% CI)
53 44 7 11
63 53 13 16
1 1 0.35 0.68
0.86 0.90 1.49 1.10
(56%) (47%) (7%) (12%)
(56%) (47%) (12%) (14%)
(0.48, (0.50, (0.55, (0.47,
Given are the number of patients with antibody titres 1:\3200 and percentage (%). Odds ratios (OR) are adjusted for age, sex.
1.54) 1.61) 4.05) 2.60)
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Table 3 Association of antibody titres to C. pneumoniae with inflammatory markers and other CAD risk markersa n
Antibody titre to IgA
Antibody titre to IgG
Gender Men Women
146 61
821 (1.09) 528 (0.80) P= 0.008
4866 (1.20) 2951 (1.11) P= 0.005
Smoking history Negative Positive
138 65
539 (0.82) 821 (1.09) P=0.008
2981 (1.15) 4866 (1.19) P =0.006
IL10 le6els Below 5 pg/ml Above 5 pg/ml
174 29
765 (1.06) 498 (1.07) P= 0.06
4537 (1.22) 2592 (0.90) P= 0.02
IgA c R IL-6c CRPc Fibrinogen ESR PAI-1c-0.01 FDP Total cholesterol Age a
0.11 0.10 0.10 0.05 −0.01 0.09 −0.04 0.05
IgG c P
R 0.11 0.17 0.14 0.4 0.9 0.2 0.5 0.5
0.06 0.12 0.03 0.02 −0.01 0.03 −0.08 0.07
P 0.4 0.07 0.7 0.8 0.8 0.7 0.3 0.3
IL-6 interleukine-6: CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; PAI-1, plasminoglen activator inhibitor-1; FDP, fibrin(ogen) degradation products given for the two-group conparison are geometric means (CV); R, Pearson correlation coefficent, c levels were logarithimically transformed before analysis.
angina, the antibody titres to C. pneumoniae were lower than in patients with levels below 5 pg/ml (geometric mean titres (25–75% range) for IgA antibodies were 498 (299 –666) versus 765 (359 – 1620), P =0.04 and for IgG antibodies 2592 (1183 – 5129) versus 4537 (1763 – 12385), P =0.02) (Table 3). Using sodium periodate-treated C. pneumoniae the association with IgA antibodies disappeared (geometric mean titre for IgA antibodies 679 (322 –1398) versus 804 (348 – 1985), P = 0.46 in cases and controls, respectively), and the association with IgG antibodies slightly decreased (median titre for IgG 2267 (1274–4654) versus 3856 (1628 – 8546), P= 0.03). No significant associations were found between levels of C. pneumoniae antibodies and CRP, age, total cholesterol, FDP, PAI-1 or ESR (Table 3). 5. Discussion In this study, we observed that the titre of antibodies to C. pneumoniae seemed somewhat higher in patients who were refractory during hospitalisation than in those who could be stabilised by medication, but this difference was not statistically significant. Also, in the logistic regression analysis a slightly, but not significantly, higher risk of refractiveness was observed with higher antibody titres to C. pneumoniae (OR 1.27 for IgG and OR 1.37 for IgA) (Table 1). Also, no relationship was observed
with the clinical course during the 18-month follow-up period. We did observe higher antibody titres to C. pneumoniae in smokers and lower titres in patients with the highest IL-10 levels, but there was no association with other inflammatory markers (fibrinogen, CRP, ESR). The prevalence of antibody titres of 1:] 3200 in the stabilised patients (53%) was similar to that in the event group in the Zutphen study (52%), and thus higher than that measured in the healthy reference group of that study (34%) [8]. This suggests that an antibody titre of 1:] 3200 to C. pneumoniae may be a risk factor not only for myocardial infarction and cardiac death, but also for clinical progression in patients with unstable angina. However, although both the Zutphen and the APRAIS study are performed in the Netherlands, this relationship needs to be studied more. C. pneumoniae enters the body via a respiratory tract infection and this can either be a primary infection, a reinfection or a reactivation. The pathogenic mechanism of C. pneumoniae in CAD consists of C. pneumoniae spreading through the body via the blood mononuclear cells, entering the vascular wall, local replication and contribution to the inflammatory process in the atherosclerotic plaque. C. pneumoniae will then die but the antigen will persist in the macrophages and this persistent antigen also contributes to the inflammatory reaction. Thus, a deregulation of the macrophage function by C. pneumoniae contributes to atherogenesis,
M.P.M. de Maat et al. / Atherosclerosis 153 (2000) 499–504
mainly through inflammatory mechanisms in the vascular system. Serum levels therefore reflect the effect on the total vascular system. The clinical symptoms of CAD, however, are caused by local disturbances. Thus, the serological measurements may not reflect accurately the local processes. Another point to consider is that, if an active C. pneumoniae infection precipitated the unstable angina, the antibody titres to C. pneumoniae on admission may not yet reflect such a recent infection. Only a few attempts to isolate C. pneumoniae from atherosclerotic lesions have been successful [23–25]. However, C. pneumoniae is detected in many blood vessels all over the body [26]. Further evidence of a causal contribution to the progression of CAD of inflammation is suggested by recent reports of a favourable response to antibiotic therapy by patients suffering from CAD [27,28]. Different methods are available for the detection of a C. pneumoniae infection. The relation with CAD has been observed using serological techniques. Other techniques that may be used for the diagnosis of C. pneumoniae infections are based on detecting C. pneumoniae, i.e. isolation of C. pneumoniae from atherosclerotic lesions, detection by PCR, immunohistochemistry, immunofluorescence and electronmicroscopy. There is little consistency between the methods that detect structural parts of the C. pneumoniae and the methods indicate the presence of C. pneumoniae (e.g. antibody titres). Furthermore, not all techniques will detect the presence of C. pneumoniae in the same lesion, since the presence of C. pneumoniae may be limited to one or just a few cells [23,29]. We observed higher IgA and IgG antibody titres to C. pneumoniae in patients with a positive history of smoking. This indicates that smokers are more susceptible to respiratory tract infections, which may contribute to their increased cardiovascular risk. The patients with the highest IL-10 levels, the main inhibitor of IL-6, had the lowest antibody titres to C. pneumoniae. Surprisingly, no relationship was observed between C. pneumoniae and IL-6 or other inflammatory markers. A possible explanation may be that the antibody titres are the result of past infections, while the plasma concentrations of inflammatory markers would reflect the acute situation. In conclusion, there was no significant association between antibody titres to C. pneumoniae and the risk of events during hospitalisation and the 18-month followup period in patients with UA.
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Antibodies to Chlamydia pneumoniae and clinical course in patients with unstable angina pectoris Moniek P.M. de Maat a,*, Jacobus M. Ossewaarde b, Peter W.H.M. Verheggen c, Cornelis Kluft a, Volkert Manger Cats c, Frits Haverkate a a
Di6ision of Vascular and Connecti6e Tissue Research, Gaubius Laboratory TNO-PG, PO Box 2215, 2301 CE, Leiden, The Netherlands b Research Laboratory for Infectious Diseases, National Institute of Public Health and the En6ironment, Biltho6en, The Netherlands c Department of Cardiology, Leiden Uni6ersity Medical Centre, Leiden, The Netherlands Received 13 September 1999; received in revised form 19 January 2000; accepted 11 February 2000
Abstract Inflammation is one of the most important mechanisms that contribute to coronary artery disease (CAD). One of the micro-organisms that is mentioned as a source of the inflammation is Chlamydia pneumoniae. In this study, we investigated the relationship between titres of IgG and IgA antibodies to C. pneumoniae and the clinical course, during hospitalisation and during an 18-month follow-up, in 211 patients admitted to hospital with unstable angina pectoris. Slightly more patients who were refractory during their hospitalisation were positive for C. pneumoniae antibodies than patients who could be stabilised by drug treatment (53 vs. 43%, for IgG and 16 vs. 11% for IgA, respectively)(n.s.). In logistic regression analysis no significant predictive values were observed for the relationship between antibody titres and clinical course. The antibody titres to C. pneumoniae were lower in the unstable angina patients who had plasma levels of interleukin-10 (IL-10) above 5 pg/ml than in the patients with levels below 5 pg/ml, and higher in smokers than in non-smokers. No associations were observed between antibody titres to C. pneumoniae and C-reactive protein (CRP), interleukin-6 (IL-6), age, total cholesterol levels, fibrin degradation products (FDP), plasminogen activator inhibitor-1 (PAI-1) and erythrocyte sedimentation rate (ESR). In conclusion, there was no significant association between antibody titres to C. pneumoniae and risk of events during hospitalisation and the 18-month follow-up period in patients admitted for unstable angina pectoris. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Unstable angina; Chlamydia pneumoniae; Inflammation; C-reactive protein; Interleukin-10
1. Introduction Inflammation is one of the most important mechanisms that contribute to the severity and the progression of coronary artery disease (CAD) [1]. Furthermore, the blood levels of the inflammatory marker fibrinogen [2] and C-reactive protein (CRP) [3,4] are associated with the risk of cardiovascular events. The association between blood levels of inflammatory markers and risk may reflect the inflammatory state of the vascular wall, but it is also possible that they are a reflection of a systemic inflammation. One cause of systemic inflammation may be chronic infec* Corresponding author. Tel.: +31-71-5181502; fax: + 31-715181904. E-mail address: [email protected] (M.P.M. de Maat).
tion. Indeed, some types of infection such as Chlamydia pneumoniae have been associated with cardiovascular risk [5]. Since 1989 C. pneumoniae has been established as a third species of the genus Chlamydia. On the basis of clinical research in the past 15 years, it is now estimated, that C. pneumoniae causes an average of 10% of cases of community-acquired pneumonia and 5% of bronchitis [6]. In the Netherlands, the prevalence of antibodies to C. pneumoniae in adults is 60–80% [7,8]. In 1988, Saikku and co-workers associated C. pneumoniae with CAD [9]. This association has been confirmed in several cross-sectional and prospective studies [8,10,11]. Mechanisms that have been suggested to underlie the association between C. pneumoniae and CAD are (a) that Chlamydia lipopolysaccharide (LPS) contributes to the formation of foam cells and (b) that
0021-9150/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 4 3 6 - 6
500
M.P.M. de Maat et al. / Atherosclerosis 153 (2000) 499–504
Chlamydia heatshock protein 60 activates macrophages [12 – 14]. In this study, we investigated the relationship between antibody titres to C. pneumoniae and the clinical course, in-hospital and during an 18-month follow-up, in 211 patients admitted to the hospital with unstable angina pectoris.
2. Patients and methods
2.1. Study population Acute phase reaction and ischaemic syndromes (APRAIS) is a prospective study on 211 patients with unstable angina pectoris, consecutively admitted to the Leiden University Medical Center. Details of the study population and recruitment have been published elsewhere [15]. Briefly, patients were included when they experienced typical angina at rest within 24 h before admission in combination with either, (1) myocardial ischaemia as reflected in ST-segment depression; or (2) electrocardiographic evidence of CAD based on prior Q-wave myocardial infarction; or (3) previous coronary angiography showing at least 50% lumen diameter narrowing in at least one large epicardial coronary artery. Patients with intercurrent inflammatory or neoplastic disease likely to be associated with acute phase responses were excluded. All patients received standard medical therapy unless contra-indicated and all decisions regarding the care of patients were made without knowledge of the biochemical study variables. The end point of the in-hospital follow-up was the occurrence of refractory unstable angina. Unstable angina was considered to be refractory if, in spite of optimal medical therapy, angina at rest reoccurred and led to the decision to perform urgent coronary angiography in order to assess the chances of successful coronary angioplasty or bypass surgery. The end point of the 18-month follow-up was a cardiac event (acute myocardial infarction, cardiac death, bypass surgery, coronary angioplasty, refractory unstable angina). The Ethics Committee of the Leiden University Medical Center approved the study protocol.
2.2. Assay procedures On admission, prior to the start of medication, venous blood was collected in glass tubes without additives and in sodium citrate (final concentration 0.011 mol/l). After complete clotting of the glass tubes, the blood was centrifuged for 30 min at 2000 × g at room temperature. The tubes with citrate were immediately placed in melting ice and centrifuged 30 min at 2000× g and 4°C. Serum and plasma were collected and stored in aliquots at −70°C until used.
IgG and IgA antibodies to C. pneumoniae have been determined using an enzyme immunoassay with minor modifications as described previously [16]. Briefly, C. pneumoniae strain TW183 was used to infect monolayers of HEp2 cells in six-well microtitre plates. After 3 days’ incubation, the antigen was purified by centrifugation through a layer of 35% sodium diatrizoate. Control antigen was prepared by the same method, but without infection of the monolayers. The antigens were used to coat microtitre plates. The genus-specific LPS antigen was destroyed in half of the microtitre plate by incubation with 1% sodium periodate in phosphatebuffered saline (PBS), pH 7.2. Destruction of the LPS results in a more specific assay [17,18]. A reference serum was included on each microtitre plate. Patient specimens were tested in twofold and the titre was calculated relative to the reference serum. CRP was measured in serum by a nephelometric method, range 0.2–1100 mg/l (N Latex CRP mono, Dade-Behring). Interleukin-6 (IL-6) was determined by a highly sensitive enzyme immunoassay, range 0.16–10 ng/l (Quantikine HS IL-6, R&D Systems, Oxon, UK). Interleukin-10 (IL-10) was determined by an enzyme immunoassay [19]. Fibrinogen was determined according to the method of Von Clauss ([20] 1957) using pooled citrated plasma (n= 25) in which the fibrinogen level was determined by a gravimetric method as reference material [21]. Erythrocyte sedimentation rate (ESR) was measured by the modified Westergren method. Plasminogen activator inhibitor (PAI-1) activity levels were determined using the method described by Verheijen et al. [22]. Fibrin(ogen) degradation products (FDP) concentrations were determined using an ELISA (Fibrinostika TDP, Organon Teknika, Turnhout, Belgium).
3. Statistical analysis Serum antibody titres to C. pneumoniae, PAI-1 and CRP were logarithmically transformed before analysis to normalise the distribution. The incidence of positive antibody titres was determined after dichotomization of the titre in 1:B 3200 and 1:] 3200. Three thousand two hundred was chosen as cut-off to exclude crossreactions with other Chlamydia species. This has also been done in the Zutphen study [8] and there it proved to be correct cut-off the use as prognostic marker. Logistic regression analysis was used to determine the odds ratio of having an event with titres 1:]3200 relative to a titre 1: B 3200, after adjustment for age and gender. The power of study to detect a relative risk of refractiveness (in-hospital) of 2.0 was 0.66 for IgG and 0.40 for IgA, for events during the 18-month
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follow-up the power was 0.66 for IgG and 0.42 for IgA, respectively. Pearson correlation coefficients were calculated to study the association between antibody titres to C. pneumoniae and other inflammatory variables. The relationship between antibody titres to C. pneumoniae and in-hospital and the 18-month follow-up was studied using logistic regression, with C. pneumoniae as a continuous variable or as a dichotomised variable (above or below a titre of 3200). A Student’s t-test on logarithmically transformed data was used to compare the levels in smokers and non-smokers. All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) for Windows, version 8.0 (SPSS Inc.).
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odds ratio of becoming refractory with a high antibody titre to C. pneumoniae on admittance was somewhat, but not significantly, higher than 1 for both IgA and IgG (1.31 (95% CI 0.55, 3.10) and 1.37 (95% CI 0.74, 2.50), respectively) (Table 1). When the relationship between antibodies to C. pneumoniae on admission and the clinical course after 18month follow-up were studied there was no significant difference in IgG antibodies in patients with or without a cardiac event but there were still somewhat more patients with increased IgA antibodies in those patients who experienced an event (Table 2). Also, in logistic regression analysis no significant predictive value of any of the baseline antibody titres to C. pneumoniae for events in the 18-month follow-up period was observed (Table 2).
4. Results
4.1. Relationship between antibody titres to C. pneumoniae and clinical course
4.2. Relationship of C. pneumoniae antibodies with inflammatory markers and other risk factors for cardio6ascular disease
Antibody titres to C. pneumoniae could be determined in 207 patients. Of these, 132 could be treated medically, and the remaining 75 patients required urgent coronary angiography because of persistent, refractory unstable angina. During the 18-month follow-up, a cardiac event occurred in 113 patients. More patients who were refractory during their hospitalisation had antibody titres to C. pneumoniae 1:] 3200 than patients who could be stabilised by drug treatment (Table 1). For IgG antibodies (both for native and for periodate-treated elementary bodies (EBs)) the excess of patients with high antibody titres was about 10%, for IgA antibodies the excess was 4%. However, these differences were not significant. The
The antibodies to native C. pneumoniae EBs on admittance to the hospital with UA were significantly higher in patients with a history of smoking than in non-smokers (geom. mean titres (25–75% range) for IgA antibodies were 821 (362–1637) versus 539 (295– 742), P= 0.006 and for IgG antibodies 4866 (1766– 12386) versus 2981 (1184–5129), P= 0.007. Using the titres to sodium periodate-treated C. pneumoniae gave similar results (geom. mean titre for IgA after periodate treatment 889 (355–2193) versus 602 (316–1158), P= 0.02 and for IgG 4131 (1734–9069) versus 2598 (1221– 4498), P= 0.005). In patients who had plasma levels of IL-10 above 5 pg/ml on admittance to the hospital with unstable
Table 1 Relationship between antibody titres to C. pneumoniae and refractiveness during hospitalisation of patients with unstable anginaa
IgA IgA IgG IgG a
(1:]3200) periodate (1:]3200) (1:]3200) periodate (1:]3200)
Stabilised (n = 132)
Refractory (n =75)
P value
OR (95% CI)
11 15 70 57
9 12 46 40
0.46 0.39 0.31 0.19
1.37 1.31 1.27 1.37
(8%) (11%) (53%) (43%)
(12%) (16%) (61%) (53%)
(0.52, (0.55, (0.70, (0.75,
3.58) 3.10) 2.31) 2.50)
Given are the number of patients with antibody titres 1:\3200 and percentage (%). Odds ratios (OR) are adjusted for age, sex.
Table 2 Relationship between antibody titres to C. pneumoniae and events during 18 months after admission in patients with unstable anginaa
IgG IgG IgA IgA a
(1:]3200) periodate (1:]3200) (1:]3200) periodate (1:]3200)
No events (n=94)
Events (n = 113)
P value
OR (95% CI)
53 44 7 11
63 53 13 16
1 1 0.35 0.68
0.86 0.90 1.49 1.10
(56%) (47%) (7%) (12%)
(56%) (47%) (12%) (14%)
(0.48, (0.50, (0.55, (0.47,
Given are the number of patients with antibody titres 1:\3200 and percentage (%). Odds ratios (OR) are adjusted for age, sex.
1.54) 1.61) 4.05) 2.60)
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Table 3 Association of antibody titres to C. pneumoniae with inflammatory markers and other CAD risk markersa n
Antibody titre to IgA
Antibody titre to IgG
Gender Men Women
146 61
821 (1.09) 528 (0.80) P= 0.008
4866 (1.20) 2951 (1.11) P= 0.005
Smoking history Negative Positive
138 65
539 (0.82) 821 (1.09) P=0.008
2981 (1.15) 4866 (1.19) P =0.006
IL10 le6els Below 5 pg/ml Above 5 pg/ml
174 29
765 (1.06) 498 (1.07) P= 0.06
4537 (1.22) 2592 (0.90) P= 0.02
IgA c R IL-6c CRPc Fibrinogen ESR PAI-1c-0.01 FDP Total cholesterol Age a
0.11 0.10 0.10 0.05 −0.01 0.09 −0.04 0.05
IgG c P
R 0.11 0.17 0.14 0.4 0.9 0.2 0.5 0.5
0.06 0.12 0.03 0.02 −0.01 0.03 −0.08 0.07
P 0.4 0.07 0.7 0.8 0.8 0.7 0.3 0.3
IL-6 interleukine-6: CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; PAI-1, plasminoglen activator inhibitor-1; FDP, fibrin(ogen) degradation products given for the two-group conparison are geometric means (CV); R, Pearson correlation coefficent, c levels were logarithimically transformed before analysis.
angina, the antibody titres to C. pneumoniae were lower than in patients with levels below 5 pg/ml (geometric mean titres (25–75% range) for IgA antibodies were 498 (299 –666) versus 765 (359 – 1620), P =0.04 and for IgG antibodies 2592 (1183 – 5129) versus 4537 (1763 – 12385), P =0.02) (Table 3). Using sodium periodate-treated C. pneumoniae the association with IgA antibodies disappeared (geometric mean titre for IgA antibodies 679 (322 –1398) versus 804 (348 – 1985), P = 0.46 in cases and controls, respectively), and the association with IgG antibodies slightly decreased (median titre for IgG 2267 (1274–4654) versus 3856 (1628 – 8546), P= 0.03). No significant associations were found between levels of C. pneumoniae antibodies and CRP, age, total cholesterol, FDP, PAI-1 or ESR (Table 3). 5. Discussion In this study, we observed that the titre of antibodies to C. pneumoniae seemed somewhat higher in patients who were refractory during hospitalisation than in those who could be stabilised by medication, but this difference was not statistically significant. Also, in the logistic regression analysis a slightly, but not significantly, higher risk of refractiveness was observed with higher antibody titres to C. pneumoniae (OR 1.27 for IgG and OR 1.37 for IgA) (Table 1). Also, no relationship was observed
with the clinical course during the 18-month follow-up period. We did observe higher antibody titres to C. pneumoniae in smokers and lower titres in patients with the highest IL-10 levels, but there was no association with other inflammatory markers (fibrinogen, CRP, ESR). The prevalence of antibody titres of 1:] 3200 in the stabilised patients (53%) was similar to that in the event group in the Zutphen study (52%), and thus higher than that measured in the healthy reference group of that study (34%) [8]. This suggests that an antibody titre of 1:] 3200 to C. pneumoniae may be a risk factor not only for myocardial infarction and cardiac death, but also for clinical progression in patients with unstable angina. However, although both the Zutphen and the APRAIS study are performed in the Netherlands, this relationship needs to be studied more. C. pneumoniae enters the body via a respiratory tract infection and this can either be a primary infection, a reinfection or a reactivation. The pathogenic mechanism of C. pneumoniae in CAD consists of C. pneumoniae spreading through the body via the blood mononuclear cells, entering the vascular wall, local replication and contribution to the inflammatory process in the atherosclerotic plaque. C. pneumoniae will then die but the antigen will persist in the macrophages and this persistent antigen also contributes to the inflammatory reaction. Thus, a deregulation of the macrophage function by C. pneumoniae contributes to atherogenesis,
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mainly through inflammatory mechanisms in the vascular system. Serum levels therefore reflect the effect on the total vascular system. The clinical symptoms of CAD, however, are caused by local disturbances. Thus, the serological measurements may not reflect accurately the local processes. Another point to consider is that, if an active C. pneumoniae infection precipitated the unstable angina, the antibody titres to C. pneumoniae on admission may not yet reflect such a recent infection. Only a few attempts to isolate C. pneumoniae from atherosclerotic lesions have been successful [23–25]. However, C. pneumoniae is detected in many blood vessels all over the body [26]. Further evidence of a causal contribution to the progression of CAD of inflammation is suggested by recent reports of a favourable response to antibiotic therapy by patients suffering from CAD [27,28]. Different methods are available for the detection of a C. pneumoniae infection. The relation with CAD has been observed using serological techniques. Other techniques that may be used for the diagnosis of C. pneumoniae infections are based on detecting C. pneumoniae, i.e. isolation of C. pneumoniae from atherosclerotic lesions, detection by PCR, immunohistochemistry, immunofluorescence and electronmicroscopy. There is little consistency between the methods that detect structural parts of the C. pneumoniae and the methods indicate the presence of C. pneumoniae (e.g. antibody titres). Furthermore, not all techniques will detect the presence of C. pneumoniae in the same lesion, since the presence of C. pneumoniae may be limited to one or just a few cells [23,29]. We observed higher IgA and IgG antibody titres to C. pneumoniae in patients with a positive history of smoking. This indicates that smokers are more susceptible to respiratory tract infections, which may contribute to their increased cardiovascular risk. The patients with the highest IL-10 levels, the main inhibitor of IL-6, had the lowest antibody titres to C. pneumoniae. Surprisingly, no relationship was observed between C. pneumoniae and IL-6 or other inflammatory markers. A possible explanation may be that the antibody titres are the result of past infections, while the plasma concentrations of inflammatory markers would reflect the acute situation. In conclusion, there was no significant association between antibody titres to C. pneumoniae and the risk of events during hospitalisation and the 18-month followup period in patients with UA.
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