Cardiovascular disease, resulting from atherosclerosis, is a leading cause of global morbidity and mortality. Genetic predisposition and classical environmental risk factors explain much of the attributable risk for cardiovascular events in populations, but other risk factors for the development and progression of atherosclerosis, which can be identified and modified, may be important therapeutic targets. Infectious agents, such as Chlamydia pneumoniae, have been proposed as contributory factors in the pathogenesis of atherosclerosis. In the present review, we consider the experimental evidence that has accumulated over the last 20 years evaluating the role of C. pneumoniae in atherosclerosis and suggest areas for future research in this field.

INTRODUCTION

Cardiovascular disease is the leading cause of morbidity and mortality in industrialized countries, and its incidence is increasing rapidly in less-developed nations [1]. Important clinical manifestations of cardiovascular disease include ACSs (acute coronary syndromes), stroke, renal failure and occlusive PVD (peripheral vascular disease). The major pathological process underlying these cardiovascular events in different vascular beds is atherosclerosis, a chronic systemic arterial inflammatory disease (reviewed in [2]). Atherosclerosis progresses over many years, influenced by the individual's genetic constitution and important ‘environmental’ risk factors, including age, sex, hyperlipidaemia, hypertension, smoking, diabetes mellitus and abdominal obesity [3].

Deposition of oxLDL [oxidized LDL (low-density lipoprotein)] within the vascular wall stimulates transmigration of monocytes into the subendothelial compartment, where these cells engulf oxLDL or glycated LDL via scavenger receptors and modify their phenotype to become activated foam-cell macrophages, resident within subendothelial ‘fatty streaks’ [4]. A dynamic interaction between inflammation (macrophages and lymphocytes) and repair responses [SMCs (smooth muscle cells) and fibroblasts] continues over many years, forming increasingly bulky and complex atherosclerotic plaques within the vessel wall, typically consisting of a fibrous cap shielding the vessel lumen from a macrophage-rich lipid core. Increased inflammatory activity within plaques may lead to weakening of the fibrous cap, with a risk of plaque rupture and exposure of the thrombogenic lipid core to blood: this is the concept of the ‘unstable plaque’ (reviewed in [5]). Rupture provokes acute haemorrhage into the plaque, followed by mural thrombosis, which may progress to cause acute vessel occlusion, either intermittently or irreversibly. In addition, atherothrombotic and platelet emboli may shower downstream to occlude the microvasculature. The nature of these pathophysiological events determines the acute ischaemic clinical syndrome, resulting, for example, in the coronary circulation, in a spectrum of presentations, including unstable angina, non-ST elevation MI (myocardial infarction) or ST elevation MI [6]. A ruptured plaque often remains unstable for several weeks, but may eventually heal via a vigorous SMC and fibroblast response to repair the fibrous cap, and this healing process may markedly increase the plaque volume. The arterial wall as a whole responds to the evolving plaque by compensatory expansion, or ‘remodelling’, a process initially described by Glagov et al. [7]; this maintains the cross-sectional area of the lumen. However, in later phases of disease, or after healed plaque rupture, arterial remodelling may no longer be sufficient and the increased plaque volume encroaches into the vessel lumen, leading to persistent arterial flow limitation and ischaemic clinical syndromes, including chronic stable angina or limb claudication.

Epidemiological studies have revealed that classic environmental risk factors could predict up to 90% of cases of established CAD (coronary artery disease) worldwide [3]. However, other risk factors that are yet to be elucidated may remain important in the initiation, progression or acute presentation of atherosclerosis. For many years, infectious agents have been thought to be associated with the disease [8]. Bacteria and viruses, such as Helicobacter pylori, cytomegalovirus and herpes simplex virus, have all been proposed to play a role, but the micro-organism most comprehensively studied is Chlamydia pneumoniae. Over the past 20 years, a variety of studies have investigated a possible link between C. pneumoniae infection and atherosclerosis. In the present review, we will consider whether the evidence from these studies supports a direct role for C. pneumoniae in the initiation, progression or clinical manifestations of atherosclerosis.

C. PNEUMONIAE

C. pneumoniae is an obligate intracellular Gram-negative bacterium that infects humans as a respiratory pathogen. It has a biphasic life-cycle, existing as either an EB (‘elementary body’) or a RB (‘reticulate body’). The EB is the extracellular infectious non-replicating form which, when internalized by a susceptible cell, differentiates into the metabolically active RB. The RB replicates, by binary fission, forming an intracellular microcolony (inclusion) and then re-differentiates, after 48–72 h, back into EB forms, which are released from the infected cell to begin another infection cycle. Under certain conditions, RBs do not re-differentiate directly into EBs, but form interim non-replicating ‘persistent bodies’, allowing the bacterium to maintain a chronic latent infection [9]. Exposure to C. pneumoniae is common, with 50% of individuals seropositive by 20 years of age and approx. 80% by 80 years of age [10].

C. pneumoniae generally causes mild upper respiratory tract infections, which range in severity from asymptomatic disease to, occasionally, severe pneumonia [11]. C. pneumoniae has been estimated to account for 10% of community-acquired pneumonia and 5% of pharyngitis, bronchitis and sinusitis [11] and, because it can maintain a chronic or latent infection, recurrence of the disease is frequent, despite treatment with antibiotics. A link between vascular disease and infection with other chlamydial species was suggested in the 1940s and 1960s (reviewed in [12]) and, with the isolation of C. pneumoniae from the respiratory tract in 1983 [13], it was speculated that this bacterium may also play a role in cardiovascular disease.

C. PNEUMONIAE IN THE PATHOGENESIS OF ATHEROSCLEROSIS

There is some evidence to suggest that C. pneumoniae could play a role in all stages of atherosclerosis, from the initial inflammatory lesion to plaque rupture. C. pneumoniae gains access to the vasculature during local inflammation of the lower respiratory tract, when the organism is disseminated around the body in blood mononuclear cells [14]. C. pneumoniae can infect vascular ECs (endothelial cells) in vitro, stimulating the secretion of pro-inflammatory cytokines and the expression of leucocyte adhesion molecules [15], which results in adhesion and trans-endothelial migration of leucocytes [16], inflammation within the vessel wall and the initial ‘response to injury’ initiating lesion formation (Figure 1).

Inflammation as a response to vascular injury

Figure 1
Inflammation as a response to vascular injury

C. pneumoniae gains entry into the vessel wall by extravasation of infected blood monocytes. C. pneumoniae infection of ECs stimulates the secretion of pro-inflammatory cytokines (e.g. TNF-α, IFN-γ and IL-6) [184] and leukocyte adhesion molecules (e.g. VCAM-1, ICAM-1 and E-selectin) [15]. This results in trans-endothelial migration of T-cells and monocytes, which differentiate into macrophages. The result is an inflammatory response within the vessel wall: an initial ‘response to injury’. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Figure 1
Inflammation as a response to vascular injury

C. pneumoniae gains entry into the vessel wall by extravasation of infected blood monocytes. C. pneumoniae infection of ECs stimulates the secretion of pro-inflammatory cytokines (e.g. TNF-α, IFN-γ and IL-6) [184] and leukocyte adhesion molecules (e.g. VCAM-1, ICAM-1 and E-selectin) [15]. This results in trans-endothelial migration of T-cells and monocytes, which differentiate into macrophages. The result is an inflammatory response within the vessel wall: an initial ‘response to injury’. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Formation of foam cells, the hallmark of early atherosclerotic lesions (fatty streaks), is the result of the uptake of oxLDL into macrophages. Native unmodified LDL does not normally play an important role in foam cell formation, due to the stringent control of LDLr (LDL receptor) expression, whereas oxLDL can be taken up by unregulated scavenger receptors. However, C. pneumoniae exposure can induce macrophages to take up increased amounts of native LDL and become foam cells, possibly due to up-regulation of LDLrs [17]. In addition, chlamydial LPS (lipopolysaccharide) and cHsp60 (chlamydial heat-shock protein 60) can induce oxidation of LDL within the neointima [18], providing a mechanism by which C. pneumoniae could enhance foam cell formation (Figure 2).

Fatty streak formation

Figure 2
Fatty streak formation

C. pneumoniae-infected ECs produce ROS (reactive oxygen species), which, together with chlamydial antigens cHsp60 and LPS, enhance the oxidation of LDL to oxLDL [18]. Exposure of macrophages to C. pneumoniae stimulates the uptake of oxLDL as well as large amounts of native LDL, which causes macrophages to develop into lipid-laden foam cells [17]. Activation of T-cells by C. pneumoniae results in the production of IFN-γ, which activates macrophages and triggers a persistent infection within macrophages [9]. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Figure 2
Fatty streak formation

C. pneumoniae-infected ECs produce ROS (reactive oxygen species), which, together with chlamydial antigens cHsp60 and LPS, enhance the oxidation of LDL to oxLDL [18]. Exposure of macrophages to C. pneumoniae stimulates the uptake of oxLDL as well as large amounts of native LDL, which causes macrophages to develop into lipid-laden foam cells [17]. Activation of T-cells by C. pneumoniae results in the production of IFN-γ, which activates macrophages and triggers a persistent infection within macrophages [9]. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

C. pneumoniae-infected ECs can secrete growth factors [19,20] which, together with cytokines and growth factors from infected macrophages and oxLDL [21], stimulate SMC proliferation and migration from the media to the intima. SMCs secrete extracellular matrix molecules, such as fibrin, proteoglycans and collagen, which contribute to the formation of a fibrous cap; in this way, C. pneumoniae infection in the vascular wall could contribute to fibrofatty plaque formation (Figure 3).

Fibrofatty plaque formation

Figure 3
Fibrofatty plaque formation

Macrophages are stimulated by cHsp60 to secrete MMPs, which degrade the internal elastic lamina [22]. Infected ECs secrete soluble factors [e.g. PDGF-B (platelet derived growth factor B) and HB-EGF (heparin-binding epidermal-growth-factor-like growth factor)], which, together with cytokines and growth factors secreted by infected macrophages, as well as oxLDL [21], stimulate SMC proliferation and migration from the media to the intima. SMCs secrete extracellular matrix molecules, such as fibrin, proteoglycans and collagen, which contributes to the formation of a fibrous cap. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Figure 3
Fibrofatty plaque formation

Macrophages are stimulated by cHsp60 to secrete MMPs, which degrade the internal elastic lamina [22]. Infected ECs secrete soluble factors [e.g. PDGF-B (platelet derived growth factor B) and HB-EGF (heparin-binding epidermal-growth-factor-like growth factor)], which, together with cytokines and growth factors secreted by infected macrophages, as well as oxLDL [21], stimulate SMC proliferation and migration from the media to the intima. SMCs secrete extracellular matrix molecules, such as fibrin, proteoglycans and collagen, which contributes to the formation of a fibrous cap. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Plaque destabilization, rupture and thrombus formation may also be influenced by C. pneumoniae infection. cHsp60 stimulates macrophages to produce MMPs (matrix metalloproteinases) [22], which weaken the plaque [23], and the up-regulation of tissue factor and PAI-1 (plasminogen activator inhibitor-1) by infected ECs and SMCs increases the likelihood of thrombosis in the event of plaque rupture [24] (Figure 4).

Plaque destabilization, rupture and thrombosis

Figure 4
Plaque destabilization, rupture and thrombosis

C. pneumoniae-activated macrophages and SMCs produce ROS (reactive oxygen species), which stimulate foam cell death and, hence, the formation of a necrotic core [185]. In response to cHsp60, macrophages and SMCs produce MMPs [22], which degrade the extracellular matrix and weaken the plaque [23]. Infected ECs and infected SMCs produce tissue factor and PAI-1, which contribute to a procoagulant state [24] and may increase the likelihood of thrombus formation when the plaque ruptures. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Figure 4
Plaque destabilization, rupture and thrombosis

C. pneumoniae-activated macrophages and SMCs produce ROS (reactive oxygen species), which stimulate foam cell death and, hence, the formation of a necrotic core [185]. In response to cHsp60, macrophages and SMCs produce MMPs [22], which degrade the extracellular matrix and weaken the plaque [23]. Infected ECs and infected SMCs produce tissue factor and PAI-1, which contribute to a procoagulant state [24] and may increase the likelihood of thrombus formation when the plaque ruptures. An animated version of this Figure is available at http://www.clinsci.org/cs/114/0509/cs1140509add.htm.

Interactions of C. pneumoniae with other risk factors for atherosclerosis

In addition to evidence that C. pneumoniae can play a role in all of the stages of atherosclerosis, there are indications that C. pneumoniae can increase the impact of the classical risk factors for atherosclerosis. As O'Conner [25] noted, understanding the interactions of C. pneumoniae with established cardiovascular risk factors may be important in clarifying the circumstances of causality.

Raised plasma levels of the acute-phase protein fibrinogen have been associated with an increased risk of CAD [26,27], because it can facilitate platelet aggregation [28] and thrombus formation [29]. Increased fibrinogen concentrations have also been associated with a poorer clinical outcome in patients with unstable CAD [30]. A number of reports have described an independent association between C. pneumoniae seropositivity and raised fibrinogen levels [3133], although no association has been observed between C. pneumoniae DNA in PBMCs (peripheral blood mononuclear cells) and fibrinogen levels [34].

A number of studies have shown an association between C. pneumoniae infection and an increase in serum triacylglycerols (triglycerides) and a decrease in HDL (high-density lipoproteins). Leinonen et al. [35] found that triacylglycerol levels were higher and HDL levels were lower in acute pneumonia caused by C. pneumoniae than in pneumonia caused by viruses and other bacteria. Similarly, a seroepidemiological study of 415 Finnish males by Laurila et al. [36] showed that serum triacylglycerol was higher and HDL-cholesterol was lower in subjects with chronic C. pneumoniae infection (persistent IgG and IgA over 3 years) than in seronegative individuals. In vitro infection of human PBMCs with C. pneumoniae dose-dependently induces the production of TNF-α (tumour necrosis factor-α), IL (interleukin)-1β, IL-6 and IFN-γ (interferon-γ) [37,38]. Administration of TNF-α to rats elevates both plasma triacylglycerol and cholesterol levels [39], with the elevation in plasma triacylglycerol attributed to stimulation of de novo hepatic fatty acid and triacylglycerol synthesis [40] and inhibition of adipose lipoprotein lipase activity [41]. Likewise, IL-1, IL-6 and IFN-γ can increase cholesterol synthesis and hepatic fatty acid synthesis, and inhibit adipose lipoprotein lipase activity [4244]. Thus chlamydial infection could have deleterious effects on serum lipid composition and, in chronic chlamydial infection, this would be compounded by the continuous production of pro-inflammatory cytokines. However, it may be erroneous to conclude that the atherogenic lipid profiles observed in seropositive individuals is a consequence of C. pneumoniae infection, as some evidence suggests that it could be part of the cause. For example, in genetically modified mice with hypercholesterolaemia [ApoE (apolipoprotein E)−/− and LDLr−/−], activation and recruitment of cytotoxic T-lymphocytes was impaired, indicating that hypercholesterolaemia has a significant suppressive effect on cellular immunity [45]. Given the importance of cell-mediated immunity in controlling C. pneumoniae infection [46], pre-existing hypercholesterolaemia could potentially increase the risk of developing a chronic C. pneumoniae infection.

An interaction between C. pneumoniae infection and smoking has been observed in several studies. Karvonen et al. [47] demonstrated, in a population-based study of 3487 subjects in Finland, that smokers were significantly more likely to be C. pneumoniae-IgG-seropositive (IgG≥1:16) and, similarly, Von Hertzen et al. [48] showed that smoking was significantly associated with higher C. pneumoniae IgA levels in 111 generally healthy smoking-discordant twins. In a study examining the relationship between C. pneumoniae infection and cardiovascular disease, Smieja et al. [49] demonstrated that current smoking was associated with a higher prevalence of circulating C. pneumoniae DNA detection in PBMCs in patients undergoing elective coronary angiography or angioplasty. Cell-mediated immunity in human twins (assessed by lymphoproliferative responses) has been shown to be lower in the smoking compared with the non-smoking twin and to be inversely correlated with humoral immunity [50], which may explain, in part, why smokers are more susceptible to C. pneumoniae infection than non-smokers. However, it is uncertain whether smoking and C. pneumoniae infections are independent risk factors for atherosclerosis. Some seroepidemiological studies reporting an association between C. pneumoniae and CAD have been criticized [51] for not reporting adequate data on smoking. Karvonen et al. [47] noted that, although the effect of current smoking was controlled for in the majority of seroepidemiological studies, ex-smokers were not distinguished from non-smokers and, given that the OR (odds ratio) for high seropositivity (IgG≥1:128) has been found to be 1.5 for smokers and 1.7 for ex-smokers, grouping never-smokers and ex-smokers together as ‘non-smokers’ could significantly reduce the calculated differential in the risk of C. pneumoniae seropositivity between smokers and non-smokers.

C. pneumoniae antibody seroprevalence rates tend to be higher in men than in women [11,53], suggesting that men are more susceptible to C. pneumoniae infection than women. Iron levels are in general higher in men than in women [54,55] and are known to be essential for developing microbial infection. Indeed, growth of C. pneumoniae in a human cell line and in Hep-2 cells was shown to be strongly inhibited by iron restriction or by the use of an iron-chelating compound [56]. The endothelial response towards chronic infections also depends on intracellular iron levels: iron further up-regulates C. pneumoniae-induced VCAM-1 (vascular cell adhesion molecule-1) expression and potentiates C. pneumoniae-induced ICAM-1 (intercellular adhesion molecule-1) expression on ECs [57]. Thus high iron levels not only predispose to C. pneumoniae infection, but could enhance the effects of C. pneumoniae on atherogenesis and may provide one mechanism whereby men have a higher prevalence of cardiovascular disease than women [58].

In summary, there is some indirect evidence that C. pneumoniae can interact with other risk factors to promote atherosclerosis. In the following sections we evaluate the evidence from a range of studies, including seroepidemiology, histopathology, animal models and clinical intervention trials, to determine whether C. pneumoniae is either necessary or sufficient to cause atherosclerosis, and its importance in the clinical consequences of atherosclerosis.

Seroepidemiological studies

The first report of a possible association between C. pneumoniae infection and atherosclerosis came in 1988 from a small cross-sectional study by Saikku et al. [59] in Finland, showing that patients with chronic stable CAD or AMI (acute MI) were significantly more likely to have increased C. pneumoniae antibodies than controls. This prompted further cross-sectional studies to investigate the association between C. pneumoniae infection and various cardiovascular events (Table 1), and a meta-analysis by Bloemenkamp et al. [60] in 2003 calculated the overall weighted OR from 29 cross-sectional studies as 2.0. An interesting observation from the meta-analysis by Bloemenkamp et al. [60], not specifically noted in that paper, is that the ORs from cross-sectional studies for patients with acute events (e.g. MI or death) were higher than those relating to patients with chronic conditions (e.g. stable CAD) (Table 2). This suggests that, although C. pneumoniae may play a role in atherosclerosis, a recently active C. pneumoniae infection (assumed by high antibody titres) may be an important factor in precipitating an acute cardiovascular event.

Table 1
Seroepidemiological studies

Studies are sorted in descending order of adjusted ORs within a type of study. ORs in bold indicate statistically significant results (P≤0.05). The power of the study to detect an OR of at least 2.0 has been calculated. *Adjustments for confounding variables (1=age+sex; 2=1+smoking; 3=2+classic cardiovascular risk factors; 4=3+socioeconomic status); †total number; ‡mean follow-up. AP, angina pectoris; athero., atherosclerosis; IA, immunoassay; EIA, enzymic IA; IC, immune complexes; TRF, time-resolved fluorimetry; ?, unknown.

Type of studyReferenceYearFirst authorCases (n)Controls (n)PowerPopulation sourceAge (years)Follow-upCase eventsTest protocolAntibody levelsSample times pointsAdj*OR
Cross-sectional                
 [591988 Saikku 40 41 25% AMI admissions (controls healthy) 45 4 weeks MI/death MIF and EIA IgA≥32 and/or IgG≥128 0 and 4 weeks 10.1 
 [1351995 Mendall 100 64 16% CAD clinic (controls healthy) 45–65 0 weeks CAD MIF IgG≥64 0 weeks 7.4 
 [591988 Saikku 30 41 23% AP admissions (controls healthy) 45 4 weeks CAD MIF IgA≥32 and/or IgG≥128 0 and 4 weeks 4.9 
 [1361999 Markus 983 − Healthy 53 0 weeks Early athero. MIF IgA≥16 0 weeks 4.0 
 [1371997 Blasi 61 61 45% AMI admission (controls healthy) 53 4 weeks MI/death MIF IgG≥16 0 and 4 weeks 3.2 
 [1382006 Elkind 239 428 97% First stroke (controls healthy) 69 0 weeks First stroke MIF IgA≥16 0 weeks 1.5 
 [1382006 Elkind 239 428 94% First stroke (controls healthy) 69 0 weeks First stroke MIF IgG≥32 0 weeks 1.2 
Prospective                
 [1392000 Siscovick 212 404 53% General population >65 3.5 years MI/death MIF IgG≥1024 Baseline (<2 years pre-) 4.2 
 [691992 Saikku 102 102 52% Dyslipidaemia 40–55 5 years MI/death MIF and IA IgA≥64 and IC present Baseline & 3–6mths pre- 2.6 
 [622002 Danesh 325 806 100% Healthy 52 16 years MI/death TRF IgA (top third of controls) Baseline 2.2 
 [691992 Saikku 102 102 52% Dyslipidaemia 40-55 5 years MI/death MIF and IA IgG≥128 and IC present 3–6mths pre- 2.2 
 [1402005 Arcari 161 161 73% AMI admissions (controls healthy) 40 10 years MI/death MIF IgA≥64 Baseline (<5 years pre-) 2.1 
 [702003 Huittenen 172 172 80% Dyslipidaemia 48 8.5 years MI/death MIF IgA≥40 Baseline & 3–6mths pre- 2.0 
 [622002 Danesh 502 1005 100% General population 52 16 years MI/death TRF IgA (top third of controls) Baseline 1.8 
 [1402005 Arcari 300 300 73% AMI admissions (controls healthy) 40 10 years MI/death MIF IgA≥64 Baseline 1.7 
 [1402005 Arcari 161 161 73% AMI admissions (controls healthy) 40 10 years MI/death MIF IgG≥256 Baseline (<5 years pre-) 1.6 
 [612000 Danesh 496 1026 100% General population 40–59 16 years MI/death TRF IgG (top third of controls) Baseline 1.2 
 [1412000 Mayr 826 – 100% Healthy 40–79 5 years Early athero. ELISA IgA (various) Baseline 1.1 
 [1422007 Westerhout 295 295 96% ACS patients 71 4 weeks MI/death MIF IgA≥16 Baseline 1.4 
 [1422007 Westerhout 138 138 76% ACS patients 75 1 year Death MIF IgA≥16 Baseline 1.3 
 [1431999 Ridker 343 343 99% Healthy 57 12 years MI/death MIF IgG (all titre bands) Baseline 1.0 
 [1422007 Westerhout 295 295 96% ACS patients 71 4 weeks MI/death MIF IgG≥32 Baseline 0.7 
 [1422007 Westerhout 138 138 76% ACS patients 75 1 year Death MIF IgG≥32 Baseline 0.6 
Meta-analyses                
 Cross-sectional                
 [602003 Bloemenkamp 4259† 4989† − Varied − Varied MI/death Various Various Various − 2.0 
 Prospective                
 [622002 Danesh 2283†  − Varied 54 11 years‡ MI/death Various IgA various Various 1.3 
 [602003 Bloemenkamp 3070† 7294† − Varied − Varied MI/death Various Various Various − 1.1 
 [612000 Danesh 3169†  − Varied 56 10 years‡ MI/death Various IgG various Various 1.2 
Type of studyReferenceYearFirst authorCases (n)Controls (n)PowerPopulation sourceAge (years)Follow-upCase eventsTest protocolAntibody levelsSample times pointsAdj*OR
Cross-sectional                
 [591988 Saikku 40 41 25% AMI admissions (controls healthy) 45 4 weeks MI/death MIF and EIA IgA≥32 and/or IgG≥128 0 and 4 weeks 10.1 
 [1351995 Mendall 100 64 16% CAD clinic (controls healthy) 45–65 0 weeks CAD MIF IgG≥64 0 weeks 7.4 
 [591988 Saikku 30 41 23% AP admissions (controls healthy) 45 4 weeks CAD MIF IgA≥32 and/or IgG≥128 0 and 4 weeks 4.9 
 [1361999 Markus 983 − Healthy 53 0 weeks Early athero. MIF IgA≥16 0 weeks 4.0 
 [1371997 Blasi 61 61 45% AMI admission (controls healthy) 53 4 weeks MI/death MIF IgG≥16 0 and 4 weeks 3.2 
 [1382006 Elkind 239 428 97% First stroke (controls healthy) 69 0 weeks First stroke MIF IgA≥16 0 weeks 1.5 
 [1382006 Elkind 239 428 94% First stroke (controls healthy) 69 0 weeks First stroke MIF IgG≥32 0 weeks 1.2 
Prospective                
 [1392000 Siscovick 212 404 53% General population >65 3.5 years MI/death MIF IgG≥1024 Baseline (<2 years pre-) 4.2 
 [691992 Saikku 102 102 52% Dyslipidaemia 40–55 5 years MI/death MIF and IA IgA≥64 and IC present Baseline & 3–6mths pre- 2.6 
 [622002 Danesh 325 806 100% Healthy 52 16 years MI/death TRF IgA (top third of controls) Baseline 2.2 
 [691992 Saikku 102 102 52% Dyslipidaemia 40-55 5 years MI/death MIF and IA IgG≥128 and IC present 3–6mths pre- 2.2 
 [1402005 Arcari 161 161 73% AMI admissions (controls healthy) 40 10 years MI/death MIF IgA≥64 Baseline (<5 years pre-) 2.1 
 [702003 Huittenen 172 172 80% Dyslipidaemia 48 8.5 years MI/death MIF IgA≥40 Baseline & 3–6mths pre- 2.0 
 [622002 Danesh 502 1005 100% General population 52 16 years MI/death TRF IgA (top third of controls) Baseline 1.8 
 [1402005 Arcari 300 300 73% AMI admissions (controls healthy) 40 10 years MI/death MIF IgA≥64 Baseline 1.7 
 [1402005 Arcari 161 161 73% AMI admissions (controls healthy) 40 10 years MI/death MIF IgG≥256 Baseline (<5 years pre-) 1.6 
 [612000 Danesh 496 1026 100% General population 40–59 16 years MI/death TRF IgG (top third of controls) Baseline 1.2 
 [1412000 Mayr 826 – 100% Healthy 40–79 5 years Early athero. ELISA IgA (various) Baseline 1.1 
 [1422007 Westerhout 295 295 96% ACS patients 71 4 weeks MI/death MIF IgA≥16 Baseline 1.4 
 [1422007 Westerhout 138 138 76% ACS patients 75 1 year Death MIF IgA≥16 Baseline 1.3 
 [1431999 Ridker 343 343 99% Healthy 57 12 years MI/death MIF IgG (all titre bands) Baseline 1.0 
 [1422007 Westerhout 295 295 96% ACS patients 71 4 weeks MI/death MIF IgG≥32 Baseline 0.7 
 [1422007 Westerhout 138 138 76% ACS patients 75 1 year Death MIF IgG≥32 Baseline 0.6 
Meta-analyses                
 Cross-sectional                
 [602003 Bloemenkamp 4259† 4989† − Varied − Varied MI/death Various Various Various − 2.0 
 Prospective                
 [622002 Danesh 2283†  − Varied 54 11 years‡ MI/death Various IgA various Various 1.3 
 [602003 Bloemenkamp 3070† 7294† − Varied − Varied MI/death Various Various Various − 1.1 
 [612000 Danesh 3169†  − Varied 56 10 years‡ MI/death Various IgG various Various 1.2 
Table 2
Cross-sectional seroepidemiology studies included in a 2003 meta-analysis [60]

The Table shows the ORs for acute and chronic events, with studies ordered by ORs. Values in bold denote results that were statistically significant (P≤0.05). The weighted average takes into account the number of cases. AP, angina pectoris; TIA, transient ischaemic attack.

ORs
YearFirst authorEventsCases (n)Controls (n)Chronic eventsAcute events
1999 Kaykov MI/AP 130 98  6.1 
1999 Kontula MI/CAD 28 68  5.7 
1997 Thomas MI/CAD 83 93  5.4 
1998 Cook Stroke/TIA 176 1518  3.8 
1999 Komer Stenosis 275 65 3.4  
1997 Blasi MI 61 61  3.2 
2000 Elkind Stroke 89 89  2.6 
1999 Sessa AP/MI 80 50  2.5 
1998 Miyashita MI 160 160  2.4 
1999 Sessa MI 98 50  2.3 
1998 Gabriel MI 146 102  1.9 
1999 Shimada Stenosis 123 29 2.3  
2000 Romeo CAD 54 49 2.0  
2000 Ammann CAD 21 42 2.0  
2000 Jantos Stenosis 489 263 1.8  
1998 Boman CAD 101 52 1.7  
1998 Anderson MI 112 197  1.6 
1998 Gabriel AP 136 102 1.5  
1999 Altman CAD/PAD 159 203 1.4  
2000 Romeo CAD 56 49 1.3  
1999 Leowaittana CAD 243 115 1.2  
1999 Cellesi Stenosis 150 56 1.2  
1998 Anderson Stenosis 219 126 1.1  
2000 Hoffmeister Stenosis 312 479 1.0  
1997 Kark MI 302 488  0.9 
1999 Cellesi Stenosis 150 49 0.8  
1999 Abdelmouttaleb Stenosis 142 74 0.8  
1999 Nobel MI/AP 58 58  0.7 
    Weighted average 1.6 2.7 
    Difference:  1.1 (P=0.03) 
ORs
YearFirst authorEventsCases (n)Controls (n)Chronic eventsAcute events
1999 Kaykov MI/AP 130 98  6.1 
1999 Kontula MI/CAD 28 68  5.7 
1997 Thomas MI/CAD 83 93  5.4 
1998 Cook Stroke/TIA 176 1518  3.8 
1999 Komer Stenosis 275 65 3.4  
1997 Blasi MI 61 61  3.2 
2000 Elkind Stroke 89 89  2.6 
1999 Sessa AP/MI 80 50  2.5 
1998 Miyashita MI 160 160  2.4 
1999 Sessa MI 98 50  2.3 
1998 Gabriel MI 146 102  1.9 
1999 Shimada Stenosis 123 29 2.3  
2000 Romeo CAD 54 49 2.0  
2000 Ammann CAD 21 42 2.0  
2000 Jantos Stenosis 489 263 1.8  
1998 Boman CAD 101 52 1.7  
1998 Anderson MI 112 197  1.6 
1998 Gabriel AP 136 102 1.5  
1999 Altman CAD/PAD 159 203 1.4  
2000 Romeo CAD 56 49 1.3  
1999 Leowaittana CAD 243 115 1.2  
1999 Cellesi Stenosis 150 56 1.2  
1998 Anderson Stenosis 219 126 1.1  
2000 Hoffmeister Stenosis 312 479 1.0  
1997 Kark MI 302 488  0.9 
1999 Cellesi Stenosis 150 49 0.8  
1999 Abdelmouttaleb Stenosis 142 74 0.8  
1999 Nobel MI/AP 58 58  0.7 
    Weighted average 1.6 2.7 
    Difference:  1.1 (P=0.03) 

Cross-sectional studies are not able to determine a causal relationship between C. pneumoniae exposure and atherosclerotic consequences, since both are observed at the same time point. However, prospective studies do enable a causal relationship to be investigated by examining infection status some years previously and are considered superior because of more stringent controls and adjustments for confounding variables. Importantly, however, meta-analyses of prospective studies, published in 2000 [61] and 2002 [62], have found either a weak or no association between increased antibody titres and cardiovascular events (Table 1).

Because the majority of seroepidemiological studies have been performed in relatively elderly populations, correlating C. pneumoniae antibody levels with infection status is problematic due to the high prevalence of C. pneumoniae seropositivity in these age groups. Furthermore, in older populations, a role for C. pneumoniae can only be investigated in the late clinical consequences of atherosclerosis, rather than a potential causal role for C. pneumoniae early in the atherosclerotic process. To address this question, Volanen et al. [63] performed a prospective study on 199 healthy children from 7 to 11 years of age. Children with persistent IgG and/or IgA seropositivity to C. pneumoniae had significantly increased aortic intima-media thickness (measured by ultrasound) at 11 years of age compared with C. pneumoniae-seronegative children and children who had been transiently C. pneumoniae seropositive, providing evidence of a possible role of persistent C. pneumoniae infection in promoting early atherosclerosis.

Previous studies have shown that the link between C. pneumoniae and CAD depends on the method chosen to measure the C. pneumoniae antibodies [6466]. Most seroepidemiological studies used the MIF (microimmunofluorescence) test and, although this is considered to be the only currently acceptable serological test [67], it is subject to substantial inter-laboratory variation [68]. Following publication of guidelines in 2001 from a workshop on the standardization of C. pneumoniae diagnostic methods [67], it has become clear that the majority of seroepidemiological studies were markedly limited because of their use of single time point sampling of either IgG or IgA antibodies to define chronic infection. The workshop concluded that there is currently no valid serological marker specifically for chronic C. pneumoniae infection and that interpretation of infection status on the basis of single titre readings was not recommended. An IgG titre≥16 was considered to indicate past exposure, but neither IgA nor any other marker was considered to be a valid indicator of persistent infection [67]. Only Saikku et al. [69], Huittenen et al. [70] and Volanen et al. [63] have reported antibody titre samples at more than one time point, which enabled a more reasonable assumption of chronic infection but, even with two samples, the possibility of re-infection, rather than chronic infection, cannot be excluded. Only Volanen et al. [63] took multiple samples (five readings at 1 year intervals), which gives more weight to the diagnosis of chronic infection in subjects who were persistently seropositive.

Defining infection by detection of C. pneumoniae DNA in PBMCs

Serological studies have provided only weak evidence, at best, for an association between C. pneumoniae seropositivity and cardiovascular events. More recent studies have evaluated whether there is an association between the presence of C. pneumoniae DNA in PBMCs and atherosclerosis. In 1998 Boman et al. [71] showed that C. pneumoniae DNA could be detected by nested PCR in PBMCs of 59% of CAD patients and 46% of middle-aged blood-donor controls, which led to the speculation that PBMC analysis may be a more useful tool than serology for the identification of C. pneumoniae carriers. C. pneumoniae in PBMCs is an indicator of current infection and can be quickly cleared once infection has resolved [72,73], although the bacteria may be harboured within monocytes in a persistent state [74].

Subsequent studies have investigated the prevalence of C. pneumoniae DNA in PBMCs in cardiovascular disease patients and controls (generally blood donors) from the general population (Table 3). In these studies, the prevalence ranged from 4–87% in patients and from 0–50% in controls. A systematic review by Smieja et al. [49] in 2002 calculated a pooled prevalence of 14.3% in cardiovascular disease patients compared with 8.5% in controls, yielding a significant OR of 2.03. However, since most studies were seeking to determine whether the detection of C. pneumoniae in PBMCs could be a useful and practical measure of infection rather than using it as a tool to determine the role of C. pneumoniae in cardiovascular disease, few of these studies adjusted for risk factors for atherosclerosis. In addition, controls, if used at all, were generally not matched to cases, nor were they screened for infection. The prevalence of infection ranged widely between studies and it is not clear whether this is attributable to real population differences or to differences in DNA extraction and PCR methodologies. In addition, there is no standardized PCR assay for C. pneumoniae, and there is poor inter-laboratory reproducibility among in-house assays [75].

Table 3
Studies of C. pneumoniae DNA in PBMCs

Studies are ordered by year. Values in bold indicate those results in which the percentage of cases with C. pneumoniae DNA in PBMCs was significantly more than the percentage of controls. The power of the trial to detect a 2-fold difference between cases and controls has been calculated. AP, angina pectoris; athero., atherosclerosis; PstI, PstI cloned fragment; nPCR, nested PCR; hemi-nPCR, hemi-nPCR; qPCR, quantitative PCR; RT-PCR, real-time PCR; t-nPCR, touchdown nPCR; ?, unknown.

DNA in PBMCs
Type of studyReferenceYearFirst authorType of casesCases (n)Type of controlsControls (n)PowerBlood (ml)Type of PCRPCR targetCasesControls
With controls              
 [711998 Boman Coronary angiography 101 Blood donors 52 100% nPCR MOMP 59% 46% 
 [1441999 Wong CAD 913 No CAD 292 90% 4–5 nPCR MOMP 9% 7% 
 [732000 Kaul CAD 28 Blood donors 19 30% 20–30 t-nPCR MOMP 46% 26% 
 [342000 Berger Vascular surgery 60 ‘Normal’ check-up patients 51 34% qPCR 16S rRNA 20% 14% 
 [1452001 Sessa Unstable angina 36 Healthy controls 42 7% hemi-nPCR pstI 17% 5% 
 [1452001 Sessa AMI 57 Healthy controls 42 7% hemi-nPCR pstI 32% 5% 
 [1462001 Maraha Aortic Aneurysm 88 Healthy controls 88 33% 16S rRNA 20% 9% 
 [1472001 Smieja Coronary angiography or angioplasty 187 Normal angiogram 21 6% nPCR MOMP 12% 10% 
 [822003 Sessa Carotid endarterectomy: symptomatic 18 Carotid endarterectomy: asymptomatic 33 43% hemi-nPCR pstI 72% 30% 
 [1482003 Tsirpanlis Haemodialysis patients with athero. 57 Haemodialysis patients without athero. 73 7% 15 nPCR RNA pol B 12% 3% 
 [1492004 Apfalter Symptomatic cerebrovascular athero. 45 Asymptomatic cerebrovascular athero. 30 7% RT-PCR MOMP 4% 7% 
 [722004 Aso ACS (AMI/AP) 88 No CAD 88 100% t-nPCR MOMP 52% 50% 
 [722004 Aso Stable CAD 164 No CAD 88 100% t-nPCR MOMP 50% 50% 
 [1502005 Podsiadly Atheroma 188 No atheroma 154 99% t-nPCR MOMP 15% 25% 
 [1512007 Wang Multivessel CAD 149 No multivessel CAD 120 13% RT-PCR MOMP 9% 3% 
Systematic review of studies with controls              
 [492002 Smieja Various (nine studies) 1763 Various 874     14.3% 8.5% 
Without controls              
 [1521999 Blasi Aortic aneurysm 41 – – – t-nPCR MOMP 46% – 
 [1532000 Maass Unstable angina 188 – – – nPCR pstI 28% – 
 [1532000 Maass AMI 50 – – – nPCR pstI 26% – 
 [1472001 Smieja Coronary angiography or angioplasty 208 – – – nPCR MOMP 12% – 
 [822003 Sessa Carotid endarterectomy 51 – – – hemi-nPCR pstI 45% – 
 [1542005 Cochrane Coronary artery disease 54 – – – nPCR MOMP 5% – 
 [1552007 Sessa Carotid atherosclerosis 30 – – – RT-PCR pstI 70% – 
 [1552007 Sessa Carotid atherosclerosis 30 – – – t-nPCR MOMP 47% – 
DNA in PBMCs
Type of studyReferenceYearFirst authorType of casesCases (n)Type of controlsControls (n)PowerBlood (ml)Type of PCRPCR targetCasesControls
With controls              
 [711998 Boman Coronary angiography 101 Blood donors 52 100% nPCR MOMP 59% 46% 
 [1441999 Wong CAD 913 No CAD 292 90% 4–5 nPCR MOMP 9% 7% 
 [732000 Kaul CAD 28 Blood donors 19 30% 20–30 t-nPCR MOMP 46% 26% 
 [342000 Berger Vascular surgery 60 ‘Normal’ check-up patients 51 34% qPCR 16S rRNA 20% 14% 
 [1452001 Sessa Unstable angina 36 Healthy controls 42 7% hemi-nPCR pstI 17% 5% 
 [1452001 Sessa AMI 57 Healthy controls 42 7% hemi-nPCR pstI 32% 5% 
 [1462001 Maraha Aortic Aneurysm 88 Healthy controls 88 33% 16S rRNA 20% 9% 
 [1472001 Smieja Coronary angiography or angioplasty 187 Normal angiogram 21 6% nPCR MOMP 12% 10% 
 [822003 Sessa Carotid endarterectomy: symptomatic 18 Carotid endarterectomy: asymptomatic 33 43% hemi-nPCR pstI 72% 30% 
 [1482003 Tsirpanlis Haemodialysis patients with athero. 57 Haemodialysis patients without athero. 73 7% 15 nPCR RNA pol B 12% 3% 
 [1492004 Apfalter Symptomatic cerebrovascular athero. 45 Asymptomatic cerebrovascular athero. 30 7% RT-PCR MOMP 4% 7% 
 [722004 Aso ACS (AMI/AP) 88 No CAD 88 100% t-nPCR MOMP 52% 50% 
 [722004 Aso Stable CAD 164 No CAD 88 100% t-nPCR MOMP 50% 50% 
 [1502005 Podsiadly Atheroma 188 No atheroma 154 99% t-nPCR MOMP 15% 25% 
 [1512007 Wang Multivessel CAD 149 No multivessel CAD 120 13% RT-PCR MOMP 9% 3% 
Systematic review of studies with controls              
 [492002 Smieja Various (nine studies) 1763 Various 874     14.3% 8.5% 
Without controls              
 [1521999 Blasi Aortic aneurysm 41 – – – t-nPCR MOMP 46% – 
 [1532000 Maass Unstable angina 188 – – – nPCR pstI 28% – 
 [1532000 Maass AMI 50 – – – nPCR pstI 26% – 
 [1472001 Smieja Coronary angiography or angioplasty 208 – – – nPCR MOMP 12% – 
 [822003 Sessa Carotid endarterectomy 51 – – – hemi-nPCR pstI 45% – 
 [1542005 Cochrane Coronary artery disease 54 – – – nPCR MOMP 5% – 
 [1552007 Sessa Carotid atherosclerosis 30 – – – RT-PCR pstI 70% – 
 [1552007 Sessa Carotid atherosclerosis 30 – – – t-nPCR MOMP 47% – 

C. pneumoniae detection within PBMCs does not necessarily imply a concomitant vascular infection. Accordingly, a number of studies have sought to determine whether there is any correlation between the presence of the bacterial DNA within atherosclerotic plaques and within PBMCs (Table 4). Results have varied widely, although, of those studies finding a moderate-to-high prevalence of C. pneumoniae in plaques (>30%), 94–100% of these C. pneumoniae-plaque-positive patients also had detectable C. pneumoniae in PBMCs. Conversely, of those studies finding a moderate-to-high prevalence of C. pneumoniae DNA in PBMCs (>40%), 71–93% of these PBMC-positive patients also had detectable C. pneumoniae in their plaques (Table 4).

Table 4
Studies investigating C. pneumoniae DNA in PBMCs and atherosclerotic plaques

The percentage of patients (proportions in parentheses) in which C. pneumoniae DNA was detected in PBMCs (PBMC+) or plaques (plaque+) is shown. In addition, the percentage of plaque+ patients that were subsequently shown to be PBMC+ [PBMC+(plaque+)] and the percentage of PBMC+ patients that were subsequently shown to be plaque+ [(plaque+(PBMC+)] are indicated.

ReferenceYearFirst authorArtery sampledPBMC+plaque+plaque+(PBMC+)PBMC+(plaque+)
[1521999 Blasi Abdominal aorta 46% (19/41) 41% (17/41) 84% (16/19) 94% (16/17) 
[342000 Berger Carotid/abdominal aorta/peripheral 20% (12/60) 20% (12/60) 42% (5/12) 42% (5/12) 
[1562002 Prager Carotid 87% (40/46) 83% (38/46) 93% (37/40) 97% (37/38) 
[822003 Sessa Carotid 45% (23/51) 35% (18/51) 78% (18/23) 100% (18/18) 
[1492004 Apfalter Carotid 4% (2/45) 0% (0/45) 0% (0/2) 0% (0/0) 
[1542005 Cochrane Carotid 5% (2/43) 33% (18/54) 0% (0/2) 0% (0/18) 
[1552007 Sessa Carotid 70% (21/30) 53% (16/30) 76% (16/21) 100% (16/16) 
[1552007 Sessa Carotid 47% (14/30) 33% (10/30) 71% (10/14) 100% (10/10) 
ReferenceYearFirst authorArtery sampledPBMC+plaque+plaque+(PBMC+)PBMC+(plaque+)
[1521999 Blasi Abdominal aorta 46% (19/41) 41% (17/41) 84% (16/19) 94% (16/17) 
[342000 Berger Carotid/abdominal aorta/peripheral 20% (12/60) 20% (12/60) 42% (5/12) 42% (5/12) 
[1562002 Prager Carotid 87% (40/46) 83% (38/46) 93% (37/40) 97% (37/38) 
[822003 Sessa Carotid 45% (23/51) 35% (18/51) 78% (18/23) 100% (18/18) 
[1492004 Apfalter Carotid 4% (2/45) 0% (0/45) 0% (0/2) 0% (0/0) 
[1542005 Cochrane Carotid 5% (2/43) 33% (18/54) 0% (0/2) 0% (0/18) 
[1552007 Sessa Carotid 70% (21/30) 53% (16/30) 76% (16/21) 100% (16/16) 
[1552007 Sessa Carotid 47% (14/30) 33% (10/30) 71% (10/14) 100% (10/10) 

Once a standardized method of blood sampling, DNA extraction and PCR analysis has been established, the detection of C. pneumoniae DNA in PBMCs could be a promising marker for active C. pneumoniae infection and, hence, a means of identifying those individuals that may benefit most from antichlamydial therapy.

Histopathological studies

The initial positive results from seroepidemiological studies encouraged investigation as to whether C. pneumoniae could be detected in atherosclerotic lesions. This was first described in 1992 by Shor et al. [76] using TEM (transmission electron microscopy) and immunocytochemistry to identify C. pneumoniae EBs in five out of seven coronary artery fatty streaks and atheromas of young men at post-mortem, but no C. pneumoniae in atheroma-free coronary arteries of controls. Several other histopathological studies reported the presence of C. pneumoniae in atherosclerotic plaques by a variety of non-culture methods. Isolation and culture of the organism from plaques to demonstrate viable bacteria has proved difficult, although it has been achieved by a few investigators (Table 5).

Table 5
Histopathological studies

Studies are shown according to the detection method used [culture, EM (electron microscopy), PCR, ICC (immunocytochemistry), IF (immunofluorescence) and ISH (in situ hybridization)]. In all studies, ‘cases’ refers to atherosclerotic (athero.) specimens. Strong evidence that C. pneumoniae is present in atherosclerotic plaques is shown in bold. Any, positive by any method, external control, obtained from a different person to the case specimen; IHC, immunohistochemistry; internal control, obtained from the same person as the case specimen; TICD, transplant-induced coronary disease.

Cases positive for C. pneumoniaeControls positive for C. pneumoniae
Study comparisonReferenceYearFirst authorType of arteryCases (n)Controls (n)Type of controlAnyCultureEMPCRICCIFISHAnyCultureEMPCRICCIFISHStrong evidence
Athero. vs. non-athero. tissue                       
 [761992 Shor Coronary External 100% (7/7)  100% (7/7)  71% (5/7)   0% (0/5)    0%   YES 
 [1571997 Chiu Carotid 76 20 External 71% (54/76)    71% (54/76)   0% (0/20)    0%   YES 
 [1581996 Ramirez Coronary 10 External 70% (7/10) 10% (1/10) 30% (3/10) 50% (5/10) 50% (5/10)  10% (1/10) 0% (0/2) 0% 0% 0% 0%  0% YES 
 [1591998 Ouchi Various 39 12 External 64% (25/39) 0% (0/24)  49% (19/39) 45% (17/38)   0% (0/12)   0% (0/2) 0% (0/12)   YES 
 [831998 Shor Various 24 11 External 67% (16/24)  77% (10/13) 58% (14/24) 60% (3/5)   9% (1/11)  0% (0/9) 9% (1/11) 0% (0/2)   YES 
 [1601995 Grayston Carotid 61 13 External 61% (37/61)   60% (3/5) 61% (37/61)   0% (0/13)    0%   YES 
 [1611993 Kuo Coronary 36 11 External 56% (20/36)  29% (6/21) 43% (13/30) 42% (15/36)   0% (0/11)   0% 0%   YES 
 [1621997 Kuo Peripheral 23 External 48% (11/23) 0% (0/26)  13% (5/39) 45% (9/20)   0% (0/8)   0% 0%   YES 
 [1631995 Kuo Coronary 18 31 External 44% (8/18)   17% (3/18) 44% (8/18)   0% (0/31)    0%   YES 
 [1642002 Shi Aorta 10 23 External 40% (4/10)  100% (2/2) 10% (1/10)   40% (4/10) 4% (1/23)   0%   4% YES 
 [1651998 Petersen Aorta 40 40 External 35% (14/40)   35% (14/40)    5% (2/40)   5%    YES 
 [1662000 Farsak Various 46 39 External 26% (12/46)   26% (12/46)    0% (0/39)   0%    YES 
 [1671997 Jackson Carotid 25 − − 75% (12/16) 4% (1/25)  24% (6/25) 50% (8/16)          YES 
 [1681998 Yamashita Carotid 19 − − 58% (11/19)    58% (11/19)          YES 
 [1691995 Campbell Coronary 38 − − 53% (20/38)  100% (2/2) 32% (12/38) 45% (17/38)          YES 
 [1701996 Blasi Aorta 51 − − 51% (26/51)   51% (26/51)           YES 
 [1711998 Davidson Coronary 60 − − 37% (22/60)   23% (14/60) 33% (20/60)          YES 
 [1722001 Johnston Carotid 46 − − 37% (17/46)   37% (17/46)           YES 
 [782001 Ong Carotid 40 40 Internal/external 0% (0/40)   0% (0/40)    0%   0%    NO 
 [791999 Jantos Coronary 50 − − 8% (4/50)   8% (4/50)   0% (0/50)        NO 
 [771996 Weiss Coronary 58 − − 2% (1/58) 0% (0/58) 0% (0/22) 2% (1/58)           NO 
 [801998 Paterson Carotid, coronary 49 − − 0% (0/49)  0% (0/49)            NO 
Athero. vs. vessel disease by other causes                       
 [841996 Muhlestein Coronary 90 12 TICD 79% (71/90)  60% (3/5)   79% (71/90)  0% (0/12)      0% YES 
Meta-analysis                       
 [1731998 Gibbs 17 studies    51%       4%       YES 
 [812002 Kalayoglu 43 studies    46%       <1%       YES 
Cases positive for C. pneumoniaeControls positive for C. pneumoniae
Study comparisonReferenceYearFirst authorType of arteryCases (n)Controls (n)Type of controlAnyCultureEMPCRICCIFISHAnyCultureEMPCRICCIFISHStrong evidence
Athero. vs. non-athero. tissue                       
 [761992 Shor Coronary External 100% (7/7)  100% (7/7)  71% (5/7)   0% (0/5)    0%   YES 
 [1571997 Chiu Carotid 76 20 External 71% (54/76)    71% (54/76)   0% (0/20)    0%   YES 
 [1581996 Ramirez Coronary 10 External 70% (7/10) 10% (1/10) 30% (3/10) 50% (5/10) 50% (5/10)  10% (1/10) 0% (0/2) 0% 0% 0% 0%  0% YES 
 [1591998 Ouchi Various 39 12 External 64% (25/39) 0% (0/24)  49% (19/39) 45% (17/38)   0% (0/12)   0% (0/2) 0% (0/12)   YES 
 [831998 Shor Various 24 11 External 67% (16/24)  77% (10/13) 58% (14/24) 60% (3/5)   9% (1/11)  0% (0/9) 9% (1/11) 0% (0/2)   YES 
 [1601995 Grayston Carotid 61 13 External 61% (37/61)   60% (3/5) 61% (37/61)   0% (0/13)    0%   YES 
 [1611993 Kuo Coronary 36 11 External 56% (20/36)  29% (6/21) 43% (13/30) 42% (15/36)   0% (0/11)   0% 0%   YES 
 [1621997 Kuo Peripheral 23 External 48% (11/23) 0% (0/26)  13% (5/39) 45% (9/20)   0% (0/8)   0% 0%   YES 
 [1631995 Kuo Coronary 18 31 External 44% (8/18)   17% (3/18) 44% (8/18)   0% (0/31)    0%   YES 
 [1642002 Shi Aorta 10 23 External 40% (4/10)  100% (2/2) 10% (1/10)   40% (4/10) 4% (1/23)   0%   4% YES 
 [1651998 Petersen Aorta 40 40 External 35% (14/40)   35% (14/40)    5% (2/40)   5%    YES 
 [1662000 Farsak Various 46 39 External 26% (12/46)   26% (12/46)    0% (0/39)   0%    YES 
 [1671997 Jackson Carotid 25 − − 75% (12/16) 4% (1/25)  24% (6/25) 50% (8/16)          YES 
 [1681998 Yamashita Carotid 19 − − 58% (11/19)    58% (11/19)          YES 
 [1691995 Campbell Coronary 38 − − 53% (20/38)  100% (2/2) 32% (12/38) 45% (17/38)          YES 
 [1701996 Blasi Aorta 51 − − 51% (26/51)   51% (26/51)           YES 
 [1711998 Davidson Coronary 60 − − 37% (22/60)   23% (14/60) 33% (20/60)          YES 
 [1722001 Johnston Carotid 46 − − 37% (17/46)   37% (17/46)           YES 
 [782001 Ong Carotid 40 40 Internal/external 0% (0/40)   0% (0/40)    0%   0%    NO 
 [791999 Jantos Coronary 50 − − 8% (4/50)   8% (4/50)   0% (0/50)        NO 
 [771996 Weiss Coronary 58 − − 2% (1/58) 0% (0/58) 0% (0/22) 2% (1/58)           NO 
 [801998 Paterson Carotid, coronary 49 − − 0% (0/49)  0% (0/49)            NO 
Athero. vs. vessel disease by other causes                       
 [841996 Muhlestein Coronary 90 12 TICD 79% (71/90)  60% (3/5)   79% (71/90)  0% (0/12)      0% YES 
Meta-analysis                       
 [1731998 Gibbs 17 studies    51%       4%       YES 
 [812002 Kalayoglu 43 studies    46%       <1%       YES 

Despite some studies finding little or no evidence for the presence of C. pneumoniae in atherosclerotic plaques [7780], a review of 43 histopathological studies concluded that C. pneumoniae could be detected in 46% of atheromatous arteries and <1% of healthy arteries [81], with focal localization of C. pneumoniae within plaques [82], so some negative studies could be explained by sampling error.

Nonetheless, detection of C. pneumoniae within atherosclerotic plaques is evidence only for association. The finding that C. pneumoniae can be detected in some early fatty streak lesions [83] could be consistent with a causal role, although the bacterium may infiltrate the early lesion only after it has begun to develop. Indeed, C. pneumoniae could be simply an ‘innocent bystander’, with a propensity to grow within diseased arterial walls. Some evidence opposing this explanation came in 1996 when it was shown that C. pneumoniae could be detected in 79% of coronary artery atheromas, but only in 4% of coronary arteries diseased as a result of chronic cardiac transplant rejection [84]. Although this indicates that C. pneumoniae does not merely have a tropism for diseased arteries in general, it is not conclusive evidence that C. pneumoniae plays a specific role in the atherosclerotic process.

Animal studies

Since neither seroepidemiological nor histopathological studies can give definitive information about a causal role of C. pneumoniae infection in atherosclerosis, various animal models have been used to address the hypothesis directly (summarized in Table 6). The New Zealand White rabbit does not develop atherosclerosis unless fed a high-cholesterol (atherogenic) diet; however, C. pneumoniae inoculation of rabbits, even fed a normal diet, produced fatty streak lesions 1 week after infection and grade III atherosclerotic lesions within 2 weeks, whereas no lesions were observed in sham-inoculated controls [85]. This provided the first direct experimental evidence that C. pneumoniae infection was sufficient to induce atherosclerosis and, importantly, it was possible to isolate viable C. pneumoniae from grade III atherosclerotic lesions.

Table 6
Experimental animal studies

*Number of weeks after the first inoculation; †C57BL/6J mice were used as controls. IFU, infection-forming units; NZW, New Zealand White; SPG, sugar/phosphate/glutamate solution; ?, unknown.

Cases
C. pneumoniaeControls
AnimalReferenceYearFirst authorAnimal strainDietnStrainDose (×106 IFU)(n)RegimenAge at start (weeks)Inoculation nInterval (weeks)Follow-up (weeks)*Findings in cases compared with controls
Rabbit                
 [851997 Fong NZW Normal 12 VR1310 10–50 Medium − 1 and 2 Grade I–II lesions 
 [931999 Fong NZW Normal 48 VR1310, AR-39 10–26 24 M. pneumoniae − 12 Grade I–III lesions 
 [931999 Fong NZW Normal 48 VR1310, AR-39 10–26 24 M. pneumoniae 12 Grade III–IV lesions 
 [1741998 Muhlestein NZW Atherogenic 20 VR1310 1–5 10 PBS 9-17 30–38 Lesion size acceleration (3.4×) 
Mouse                
 Normocholesterolaemic                
 [941999 Hu LDLr−/− Normal 16 AR-39 5–10 14 SPG 4-5 42 No effect 
 [862000 Blessing C57BL/6J Normal 40 AR-39 30 24 PBS 4, 7 and 11 Inflammatory changes in aorta 
 [1752001 Burian BALB/C Normal 10 TWAR 0.02–0.04 McCoy 6-8 Inflammatory changes in aorta 
 Hypercholesterolaemic                
 [872001 Blessing C57BL/6J Atherogenic 37 AR-39 30 44 PBS 16 Lesion size acceleration (3.3×) 
 [1762001 Burnett ApoE−/− Normal 10 10 10 Lesion size acceleration (1.7×) 
 [1771999 Moazed ApoE−/− Normal 23 AR-39 30 22 PBS 8 and 12 Lesion size acceleration (1.6x) 
 [941999 Hu LDLr−/− Atherogenic 16 AR-39 5–10 14 SPG 4-5 42 Lesion size acceleration (1.5×) 
 [941999 Hu LDLr−/− Atherogenic 16 AR-39 5–10 10 C. trachomatis 4-5 42 Lesion size acceleration (1.5×) 
 [1782000 Liu LDLr−/− Atherogenic 11 AR-39 5–10 SPG 4-6 12 28 Lesion size acceleration (1.3×) 
 [1792000 Liuba ApoE−/− Normal 24 IOL-207 0.4 21 PBS 2 and 6 Endothelial dysfunction 
 [1802003 Liuba ApoE−/− Normal IOL-207 0.2 10 Endothelial dysfunction 
 [922003 Ezzahiri LDLr/ApoE−/− Normal 16 TWAR 2043 5–50 16 SPG 16 24 Reduced fibrous cap area 
 [902001 Aalto-Setala ApoE−/− Atherogenic 16 K7 0.1 24 SPG 3 or 4 1 or 3–4 10 or 18 No effect 
 [912001 Caligiuri ApoE−/− Normal 43 K6 C57BL/6J† 6-8 18 22 No effect 
 Atherogenic diet started after infection                
 [882002 Blessing C57BL/6J Atherogenic 29 AR-39 30 28 PBS 22 No effect 
Cases
C. pneumoniaeControls
AnimalReferenceYearFirst authorAnimal strainDietnStrainDose (×106 IFU)(n)RegimenAge at start (weeks)Inoculation nInterval (weeks)Follow-up (weeks)*Findings in cases compared with controls
Rabbit                
 [851997 Fong NZW Normal 12 VR1310 10–50 Medium − 1 and 2 Grade I–II lesions 
 [931999 Fong NZW Normal 48 VR1310, AR-39 10–26 24 M. pneumoniae − 12 Grade I–III lesions 
 [931999 Fong NZW Normal 48 VR1310, AR-39 10–26 24 M. pneumoniae 12 Grade III–IV lesions 
 [1741998 Muhlestein NZW Atherogenic 20 VR1310 1–5 10 PBS 9-17 30–38 Lesion size acceleration (3.4×) 
Mouse                
 Normocholesterolaemic                
 [941999 Hu LDLr−/− Normal 16 AR-39 5–10 14 SPG 4-5 42 No effect 
 [862000 Blessing C57BL/6J Normal 40 AR-39 30 24 PBS 4, 7 and 11 Inflammatory changes in aorta 
 [1752001 Burian BALB/C Normal 10 TWAR 0.02–0.04 McCoy 6-8 Inflammatory changes in aorta 
 Hypercholesterolaemic                
 [872001 Blessing C57BL/6J Atherogenic 37 AR-39 30 44 PBS 16 Lesion size acceleration (3.3×) 
 [1762001 Burnett ApoE−/− Normal 10 10 10 Lesion size acceleration (1.7×) 
 [1771999 Moazed ApoE−/− Normal 23 AR-39 30 22 PBS 8 and 12 Lesion size acceleration (1.6x) 
 [941999 Hu LDLr−/− Atherogenic 16 AR-39 5–10 14 SPG 4-5 42 Lesion size acceleration (1.5×) 
 [941999 Hu LDLr−/− Atherogenic 16 AR-39 5–10 10 C. trachomatis 4-5 42 Lesion size acceleration (1.5×) 
 [1782000 Liu LDLr−/− Atherogenic 11 AR-39 5–10 SPG 4-6 12 28 Lesion size acceleration (1.3×) 
 [1792000 Liuba ApoE−/− Normal 24 IOL-207 0.4 21 PBS 2 and 6 Endothelial dysfunction 
 [1802003 Liuba ApoE−/− Normal IOL-207 0.2 10 Endothelial dysfunction 
 [922003 Ezzahiri LDLr/ApoE−/− Normal 16 TWAR 2043 5–50 16 SPG 16 24 Reduced fibrous cap area 
 [902001 Aalto-Setala ApoE−/− Atherogenic 16 K7 0.1 24 SPG 3 or 4 1 or 3–4 10 or 18 No effect 
 [912001 Caligiuri ApoE−/− Normal 43 K6 C57BL/6J† 6-8 18 22 No effect 
 Atherogenic diet started after infection                
 [882002 Blessing C57BL/6J Atherogenic 29 AR-39 30 28 PBS 22 No effect 

C. pneumoniae inoculation of C57BL/6 (‘wild-type’) mice, fed a standard rodent chow, resulted in inflammatory changes in the aorta, but, in contrast with rabbits, did not induce clear-cut atherosclerosis [86], indicating that C. pneumoniae is alone unable to cause atherosclerosis in healthy wild-type mice. However, inoculation of C57BL/6 mice fed a high-fat diet did result in the acceleration of hypercholesterolaemia-induced atherosclerosis [87], but not if the atherogenic diet was started after the infection [88]. This suggests that, in the mouse, an active C. pneumoniae infection must occur in the setting of hypercholesterolaemia for it to influence the progression of atherosclerosis.

Establishment of a chronic infection appears important for the atherogenic properties of C. pneumoniae in mice, since persistence of the bacteria in the aorta was observed only after repeated inoculations [89]. Indeed, the inability of the studies by Aalto-Setala et al. [90] and Caligiuri et al. [91] to observe an effect of C. pneumoniae on atherosclerosis could be explained by a failure to establish a chronic infection, due either to use of a rapidly cleared strain [90] or because of a long interval between inoculations [91]. In addition to a role in the initiation and progression of atherosclerosis, studies in ApoE−/− and LDLr−/− mice (fed standard chow) have shown that C. pneumoniae infection can promote plaque instability by decreasing the fibrous cap area of plaques [92].

An important limitation of many of the animal studies is that saline sham inoculations were used as controls (Table 6). Accordingly, those studies could not definitively establish whether a generalized immune response to infection or whether C. pneumoniae infection itself was responsible for the effects observed. However, one rabbit study used Mycoplasma pneumoniae [93] and one study in LDLr−/− mice used Chlamydia trachomatis [94] as controls for C. pneumoniae infection, so these experiments were able to attribute the atherogenic effects specifically to C. pneumoniae.

Studies in rabbits and mice show that acute C. pneumoniae infection is alone sufficient to initiate atherosclerosis, at least in rabbits, and for chronic infection to accelerate hypercholesterolaemia-induced atherosclerosis and adverse changes in plaque morphology. However, although these experimental studies demonstrate proof-of-principle and possible mechanisms of action, we should exercise caution in translating these results to humans because of potential species differences.

Clinical intervention trials

If active C. pneumoniae has a role in precipitating acute cardiovascular events, one would expect effective antibiotic treatment to reduce the incidence of those events. Retrospective case-control studies evaluating routine clinical tetracycline or quinolone antibiotic use in the years prior to an acute cardiac event have produced conflicting results [95,96]. However, patients in those studies had not been treated specifically for C. pneumoniae infection. The first secondary prevention clinical trial to be performed, in 1997, was a pilot study by Gupta et al. [97], in which 60 male post-MI patients, persistently seropositive for C. pneumoniae (IgG≥1:64 on two occasions 3 months apart), were randomized to receive a 3- or 6-day course of 500 mg/day azithromycin or placebo. After 18 months follow-up, the OR for cardiovascular events (a composite of MI, unstable angina, revascularization or cardiovascular death) was 5-fold lower in patients receiving antibiotic treatment compared with placebo [OR, 0.2 (95% confidence interval, 0.05–0.8); P=0.03). However, 20 patients who had not been randomized for the treatment phase of the study were included in the OR analysis, only 12 of whom had persistently elevated C. pneumoniae IgG antibodies. The authors [97] included these patients retrospectively with the randomized placebo group on the basis that there was no significant difference in cardiovascular risk factors or events between them and the randomized placebo group. This analytical approach has since been criticised and, indeed, when these 20 non-randomized patients were excluded from the original analysis, within the context of a meta-analysis, the OR in favour of antibiotic therapy was no longer significant [98]. Even considering the original analysis reported in the study by Gupta et al. [97], the marked reduction in cardiovascular events after one or two short courses of antibiotics is probably due to a type 1 statistical error based on a total of only 14 events [97]. Another pilot secondary prevention trial, the ROXIS (Roxithromycin in Ischemic Syndromes) study [99,100], randomized 202 patients with ACS to receive a 30-day course of 150 mg of roxithromycin or placebo twice a day. After 1 month follow-up, there was a significant reduction in adverse cardiovascular events (a composite of severe recurrent angina, MI and ischaemic death) in the antibiotic treatment group [two out of 102 patients compared with nine out of 100; P=0.032] [99], but at 6 months the reduction was no longer significant [100]. However, further adequately powered studies with longer follow up [101106] and meta-analyses [98,107] have shown no evidence of a treatment benefit in either stable coronary syndrome or ACS patients (Table 7). In the most recent large-scale trial, the CLARICOR study, 4373 stable CAD patients were randomized to receive a 2-week course of 500 mg of clarithromycin or placebo/day. After 3-years follow-up, there was no significant difference between the two groups in the combined end point of death, unstable angina and MI, but, importantly and unexpectedly, cardiovascular mortality was significantly higher in those patients that had received antibiotic treatment [104].

Table 7
Clinical intervention trials

The power of the trial to detect a 30% reduction in events with treatment has been calculated. An OR in bold indicates statistically significant results (P≤0.05). All trials were randomized, double-blinded and placebo-controlled, enrolling patients diagnosed with CAD. *Adjustments for confounding variables (1=age+sex; 2=1+smoking; 3=2+classic cardiovascular risk factors; 4=3+socioeconomic status); †OR of death, unstable angina or MI; ‡Cases/controls. antiCp, antibiotics effective against C. pneumoniae; macro., macrolides; nonantiCp, antibiotics ineffective against C. pneumoniae; quin., quinolones; ?, unknown. ACADEMIC, Azithromycin in Coronary Artery Disease: Elimination of Myocardial Infection with Chlamydia; WIZARD, Weekly Intervention With Zithromax Against Atherosclerotic-Related Disorders; ACES, Azithromycin and Coronary Events Study; CLARIFY, Clarithromycin in Acute Coronary Syndrome Patients in Finland; STAMINA, South Thames Trial of Antibiotics in Myocardial Infarction and Unstable Angina; ANTI-BIO, ANTIBIOtic therapy after an AMI; PROVE-IT, Pravastatin or Atorvastatin Evaluation and Infection Therapy.

PreventionTrialReferenceYearFirst authorMean age (years)Serological inclusion criterianPowerTreatmentDuration of treatmentFollow-upAdjustment*OR†Effect
Secondary prevention               
 Stable CAD patients               
  [971997 Gupta 60 IgG≥64 60 13% Azithromycin 3 or 6 days 18 months 0.2 Positive 
 ACADEMIC [1012000 Muhlestein 64 IgG≥16 302 33% Azithromycin 3 months 2 years 0.9 Negative 
 WIZARD [1022003 O'Connor 62 IgG≥16 7747 100% Azithromycin 12 weeks 2.5 years 1.0 Negative 
 ACES [1032005 Grayston 65 None 4012 100% Azithromycin 1 year 3.9 years 1.0 Negative 
 CLARICOR [1042006 Jespersen 65 None 4373 99% Clarithromycin 2 weeks 3 years 1.2 Negative 
 ACS patients               
 ROXIS [1001999 Gurfinkel 61 None 202 15% Roxithromycin ∼24 days 1 and 6 months 0.2 (1 month)/0.6 (6 months) Positive 
 CLARIFY [1812002 Sinisalo 64 None 148 31% Clarithromycin 85 days 3 years 0.5 Positive 
 STAMINA [1822002 Stone 66 None 325 55% Azithromycin or Amoxicillin 1 week 1 year 0.6 Positive 
 ANTI-BIO [1052003 Zahn 60 None 872 76% Roxithromycin 6 weeks 1 year − 1.2 Negative 
 PROVE-IT [1062005 Cannon 58 None 4162 100% Gatifloxacin 2 weeks and then 10 weeks 2 years 1.0 Negative 
Meta-analysis of secondary prevention               
  [982004 Wells 64 None 11015 − Macrolides − <3 years − 0.9 Negative 
  [1072005 Andraws 62 None 19217 − Macrolides − <3 years − 1.0 Negative 
Primary prevention               
 Retrospective case control  [951999 Meier <75 None 3315/13139‡ 100% Antibiotic use in preceding 3 years   0.5 (quin.)0.9 (macro.) Positive 
  [961999 Jackson 67 None 1796/4882‡ 100% Antibiotic use in preceding 5 years   1.1 Negative 
 Prospective nested case control               
  [1832003 Brassard 78 None 1047/5235‡ 100% Antibiotics  5 years 0.7 (antiCp)  
             1.2 (nonantiCp) Negative 
PreventionTrialReferenceYearFirst authorMean age (years)Serological inclusion criterianPowerTreatmentDuration of treatmentFollow-upAdjustment*OR†Effect
Secondary prevention               
 Stable CAD patients               
  [971997 Gupta 60 IgG≥64 60 13% Azithromycin 3 or 6 days 18 months 0.2 Positive 
 ACADEMIC [1012000 Muhlestein 64 IgG≥16 302 33% Azithromycin 3 months 2 years 0.9 Negative 
 WIZARD [1022003 O'Connor 62 IgG≥16 7747 100% Azithromycin 12 weeks 2.5 years 1.0 Negative 
 ACES [1032005 Grayston 65 None 4012 100% Azithromycin 1 year 3.9 years 1.0 Negative 
 CLARICOR [1042006 Jespersen 65 None 4373 99% Clarithromycin 2 weeks 3 years 1.2 Negative 
 ACS patients               
 ROXIS [1001999 Gurfinkel 61 None 202 15% Roxithromycin ∼24 days 1 and 6 months 0.2 (1 month)/0.6 (6 months) Positive 
 CLARIFY [1812002 Sinisalo 64 None 148 31% Clarithromycin 85 days 3 years 0.5 Positive 
 STAMINA [1822002 Stone 66 None 325 55% Azithromycin or Amoxicillin 1 week 1 year 0.6 Positive 
 ANTI-BIO [1052003 Zahn 60 None 872 76% Roxithromycin 6 weeks 1 year − 1.2 Negative 
 PROVE-IT [1062005 Cannon 58 None 4162 100% Gatifloxacin 2 weeks and then 10 weeks 2 years 1.0 Negative 
Meta-analysis of secondary prevention               
  [982004 Wells 64 None 11015 − Macrolides − <3 years − 0.9 Negative 
  [1072005 Andraws 62 None 19217 − Macrolides − <3 years − 1.0 Negative 
Primary prevention               
 Retrospective case control  [951999 Meier <75 None 3315/13139‡ 100% Antibiotic use in preceding 3 years   0.5 (quin.)0.9 (macro.) Positive 
  [961999 Jackson 67 None 1796/4882‡ 100% Antibiotic use in preceding 5 years   1.1 Negative 
 Prospective nested case control               
  [1832003 Brassard 78 None 1047/5235‡ 100% Antibiotics  5 years 0.7 (antiCp)  
             1.2 (nonantiCp) Negative 

There are a number of limitations common to these clinical trials. All were performed without a consensus on suitable antibiotics, optimal dosage or treatment duration, so there was wide variation in treatment regimens, using different antibiotic classes for durations ranging from a few days to 1 year. The different life-cycle forms of C. pneumoniae are not equally susceptible to antibiotics and, since EBs can remain viable for months [108], prolonged treatment would be needed to prevent reactivation of infection. However, chronic antibiotic treatment may induce antibiotic resistance, as documented for mycobacteria [109]. Although antibiotic-resistant strains of C. trachomatis have been identified [110112], antibiotic resistance has not yet been unequivocally described in C. pneumoniae. Roblin et al. [113] reported that seven out of 46 patients with respiratory C. pneumoniae infection still had positive cultures after antibiotic treatment and, in two of these patients, the post-treatment MIC (lowest antibiotic concentration at which no inclusions were seen) had increased 4-fold. However, these MIC values were still within the range considered to indicate susceptibility to the antibiotic, and it was not clear whether the increase in MIC was an isolated event or whether it was suggestive of possible development of resistance. Importantly, subinhibitory concentrations of antibiotics are able to induce the persistent form of C. pneumoniae [114], which is more resistant to antibiotics.

The patients recruited into the secondary prevention trials already had advanced atherosclerosis and it is unknown whether antibiotic treatment soon after infection, and much earlier in the course of atherosclerosis, might alter long-term outcomes. Animal studies indicate that antibiotics are effective only if treatment is started soon after C. pneumoniae infection [115,116], raising the possibility that antibiotics must be started before the bacteria have become chronically established in the lesion.

Taken together, the balance of evidence from clinical antibiotic intervention trials is disappointing and does not support the treatment of C. pneumoniae infection in late established atherosclerosis.

C. pneumoniae vaccine

Considerable progress towards developing a C. pneumoniae vaccine has been made in the past decade, but a vaccine that offers long-lasting immunity in humans is not yet available. The sequencing of the C. pneumoniae genome has been helpful in identifying antigens that may elicit protective immunity [117,118]. A key challenge is the need for a vaccine to confer stronger immunity than natural infection, since re-infection in humans after previous C. pneumoniae exposure is common [53]. As an obligate intracellular pathogen, C. pneumoniae may escape humoral immune defences. In contrast, cellular immune responses, in particular CD8+ T-cells, appear to be necessary for protection against primary infection, as well as re-infection [46,119], so a successful C. pneumoniae vaccine should ideally induce a vigorous cellular immune response.

A promising approach is DNA immunization, whereby a plasmid DNA encoding the antigen of choice is injected into muscle, where synthesis of the antigen takes place. An advantage of this approach is that, in addition to coding for the antigen, plasmid DNA itself constitutes a powerful T-helper cell adjuvant due to immunostimulatory motifs [120]. A number of studies have tested DNA vaccines in animal models, encoding for several C. pneumoniae proteins, including the MOMP (major outer membrane protein) [121,122], Omp2 [121] and cHsp60 [121,123]. However, these vaccines were able to induce only partial protective immunity against C. pneumoniae challenge.

In 2007, Thorpe et al. [124] reported studies using a recombinant protein vaccine that induced an immune response able to completely eliminate C. pneumoniae infection in mice. The antigen, LcrE, a component of the chlamydial type III secretion system, induced CD4+ and CD8+ T-cell activation, type I cytokine secretion and neutralizing antibodies, and reduced the day 7 lung bacterial burden to a level undetectable by conventional immunohistochemistry and barely detectable by PCR amplification. In contrast, the challenge inoculum remained present in unvaccinated mice and in those vaccinated with other chlamydial proteins. Thorpe et al. [124] proposed that, by the induction of CD8+ T-cells and IFN-γ, this vaccine may also be effective against chronic infection, although further studies are needed.

In 2000, the Vaccine Development and Field Trials Workshop Group considered the feasibility of conducting clinical trials for a C. pneumoniae vaccine [125]. The Workshop discussed whether a C. pneumoniae vaccine should be considered for primary prevention in non-infected subjects or as a therapy to treat established chronic infection. Seroepidemiological studies suggest that children begin to seroconvert at approx. 5 years of age [10]; accordingly, for prophylactic use, the vaccine would need to be administered in infancy. A vaccine for therapeutic use in older subjects would be difficult to target and evaluate since appropriate markers for identifying patients with chronic infection are not yet available. There is a paradox in that the rationale for a C. pneumoniae vaccine to reduce cardiovascular disease remains uncertain, yet if prevention of infection by vaccination is eventually shown to reduce cardiovascular disease, this would be powerful evidence of a contributory role for C. pneumoniae in human atherosclerosis. However, the required safety and efficacy trials of any vaccine delivered early in life to modulate progression of atherosclerosis would take decades to complete. Vaccine trials to reduce acute C. pneumoniae respiratory tract infection in children, as opposed to atherosclerosis later in adult life, would have the advantage of much earlier efficacy end points, but, as acute respiratory infection is rarely severe, the clinical justification for such a vaccine strategy is questionable.

DOES C. PNEUMONIAE CAUSE ATHEROSCLEROSIS?

Evidence from a variety of approaches points to a pathogenic role for C. pneumoniae in atherosclerosis, but it is difficult to attribute causality to a common infectious agent in a highly prevalent multifactorial disease. To establish a causal relationship between a pathogen and a disease, Koch's postulates have classically been used [126] (Table 8). Koch applied these postulates to demonstrate that Mycobacterium tuberculosis causes tuberculosis and that Bacillus anthracis causes anthrax. However, Koch himself noted that there were a number of pathogens that were clearly responsible for certain diseases, but which did not fulfil all of his rigorous postulates. Detecting a pathogen in every case of the disease is not realistic in the case of multifactorial diseases, such as atherosclerosis (Postulate 1). Depending on the host, its effects are not always pathogenic (Postulate 2) and, for many pathogens, isolation and pure culture is challenging (Postulate 3).

Table 8
Summary of Koch's postulates (1884)

*Koch did not originally specify this criterion, as published by his colleague Friedrich Loeffler [126], but it was later included in his postulates.

PostulateDescription
The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease. 
The parasite occurs in no other disease as a fortuitous and non-pathogenic parasite. 
After being fully isolated from the body and repeatedly grown in pure culture, the parasite can induce the disease anew. 
The parasite should be able to be re-isolated from the experimentally infected host* 
PostulateDescription
The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease. 
The parasite occurs in no other disease as a fortuitous and non-pathogenic parasite. 
After being fully isolated from the body and repeatedly grown in pure culture, the parasite can induce the disease anew. 
The parasite should be able to be re-isolated from the experimentally infected host* 

As an alternative to Koch's postulates, Hill's criteria, proposed in 1965 [127], can be used to distinguish between disease causation and association, and allow a range of evidence to be appraised within a formal structure (Table 9). The strength of the association is related to the likelihood of a causal relationship [127]. Histopathological studies provide evidence for a moderately strong association between C. pneumoniae and atherosclerosis, in which C. pneumoniae can be detected, on average, in 46% of atheromatous arteries, but in <1% of healthy arteries [81]. Apart from a minority of histopathological studies [7780], there has been a high degree of consistency in the association between C. pneumoniae and arterial atheromatous lesions (Table 5). Similarly, animal studies have consistently demonstrated atherosclerotic effects of C. pneumoniae infection (Table 6). However, publication bias may reduce the number of negative studies, leading to a false impression of a strong and consistent association. The specificity criterion maintains that exposure to C. pneumoniae leads to only atherosclerosis and that atherosclerosis results only from C. pneumoniae infection. This is clearly not the case, but a lack of specificity in a multifactorial disease, such as atherosclerosis, does not in itself exclude a causal relationship. In the temporality criterion, exposure to C. pneumoniae must precede the onset of atherosclerosis. This has been demonstrated in animal studies in which fatty streaks, the earliest manifestation of atheroma, can be observed just 1 week after C. pneumoniae inoculation [93]. In humans, C. pneumoniae has been detected in histopathological studies of early fatty streak lesions [83], and persistent seropositivity has been associated with increased aortic intima-media thickness in children [63], but as yet there is no direct clinical evidence of C. pneumoniae infection preceding atherosclerosis. The biological gradient criterion is the requirement for a dose–response relationship. Some evidence for this has come from animal studies in which grade III–IV lesions were observed in rabbits inoculated with C. pneumoniae three times, whereas only grade I–III lesions were observed in those inoculated once [93]. Experimental studies in animals and using in vitro cellular systems have provided plausible mechanisms by which C. pneumoniae could cause atherosclerosis at all stages, from the initial inflammatory lesion to plaque rupture (Figures 1–4). The coherence criterion requires that the proposed causal relationship does not contradict existing theory or knowledge of atherosclerosis. In this respect, atherosclerosis is established as a chronic inflammatory process in response to injury [2], coherent with a proposed role for chronic C. pneumoniae infection in the vascular wall. The experiment criterion is to some extent satisfied by findings from animal models, in which treatment with antibiotics has been shown to prevent the development of atherosclerosis in response to C. pneumoniae inoculation [115]. However, in human clinical trials of antibiotic therapy, the most recent meta-analysis has indicated no overall beneficial effect for reasons discussed above [107]. If C. pneumoniae immunization becomes a clinical reality, an eventual effect on progression of atherosclerosis or its late clinical consequences would provide powerful evidence of causation rather than mere association. Finally, a causal role for C. pneumoniae infection may be plausible by analogy with the recently described association between chronic HIV infection and accelerated atherosclerosis (reviewed in [128]), possibly mediated through alterations in the cholesterol metabolism of HIV-infected macrophages in the arterial wall [129]. Taken together, C. pneumoniae satisfies most, but not all, of Hill's criteria as a causal factor in atherosclerosis.

Table 9
Summary of Hill's criteria (1965)
Criterion
Strength How strong is the association? 
Consistency Has the observed association been repeatedly observed by different persons, in different places, circumstances and times? 
Specificity Is the outcome unique to the exposure? 
Temporality Does exposure precede the outcome? 
Biological gradient As the level of exposure increases, does the rate of disease also increase? 
Plausibility Is a causal relationship biologically feasible? 
Coherence Is a causal association compatible with the generally known facts of the natural history and biology of the disease? 
Experiment Do interventions that modify exposure modify the outcome? 
Analogy Has a similar causal relationship been observed with another exposure and/or disease? 
Criterion
Strength How strong is the association? 
Consistency Has the observed association been repeatedly observed by different persons, in different places, circumstances and times? 
Specificity Is the outcome unique to the exposure? 
Temporality Does exposure precede the outcome? 
Biological gradient As the level of exposure increases, does the rate of disease also increase? 
Plausibility Is a causal relationship biologically feasible? 
Coherence Is a causal association compatible with the generally known facts of the natural history and biology of the disease? 
Experiment Do interventions that modify exposure modify the outcome? 
Analogy Has a similar causal relationship been observed with another exposure and/or disease? 

FUTURE DIRECTIONS

Where should future research in this field be focused? Further seroepidemiological studies in older subjects are probably unwarranted because of the inability to distinguish between a previous and persistent infection. Prospective serological studies of children and young adults, combined with non-invasive assessment of vascular structure and function, may provide important information on the role of C. pneumoniae in the initiation of atherosclerosis. Similarly, further histopathological studies in older subjects are not needed, since numerous groups have demonstrated the presence of C. pneumoniae in advanced plaques, but studies investigating the prevalence of the bacteria in early lesions of younger adults would be informative, although such samples are not commonly available.

Determining the presence of C. pneumoniae DNA in PBMCs offers the potential for identifying currently infected patients, once a standardized PCR assay has been established. In particular, the ability to genetically distinguish C. pneumoniae variants with vascular tropisms (for example, on the basis of tyrosine/tryptophan permease gene copy number [130]) may enable individuals at higher risk of atherosclerosis to be identified.

Further large scale clinical trials of established antibiotics, such as azithromycin, are now unlikely to be conducted in older patients with late stage atherosclerosis. However, a new class of antibiotics has been developed, the ansamycins, which inhibit the bacterial DNA-dependent RNA polymerase [131]. One of these, benzoxazinorifamycin (rifalazil), is 10–1000-fold more active than azithromycin against C. pneumoniae [132], has a long plasma and tissue half-life, permitting once-weekly treatment, and does not interact with the cytochrome P450 system. This antibiotic is currently undergoing clinical evaluation for treatment of C. pneumoniae in atherosclerosis. The PROVIDENCE-1 (Prospective Evaluation of Rifalazil Effect On Vascular Symptoms of Intermittent Claudication and Other Endpoint) trial is a phase III multinational randomized double-blind placebo-controlled trial of rifalazil in C. pneumoniae-seropositive patients with PAD (peripheral artery disease). Preliminary negative results were reported in abstract form at the American Heart Association Scientific Sessions, November 2007 [132a]. The RESTORE-IT (Randomized Evaluation of Short-Term Rifalazil Treatment on Carotid Atherosclerosis and Intima Media Thickness) trial is a phase II multicentre randomized double-blind placebo-controlled trial recruiting C. pneumoniae-seropositive patients with PVD, cerebrovascular disease or CAD. Patients will receive rifalazil or placebo once a week for 12 weeks and will be followed for 18 months with serial MRI (magnetic resonance imaging) and ultrasound to determine carotid artery intima-media thickness (at 6, 12 and 18 months). Results are expected in late 2008.

In addition to treatment of patients with established atherosclerosis, there may be a rationale for antibiotic therapy in young adults, but this will require optimization of therapeutic regimens to eliminate persistent or latent vascular infection, and development of sensitive and specific tests for determining C. pneumoniae infection and confirming its successful clearance.

It may be worthwhile targeting mechanisms by which C. pneumoniae could induce or exacerbate vascular inflammation. For example, C. pneumoniae can stimulate platelet activation and aggregation [133], enhancing thrombotic risk in acute vascular syndromes. Kalvegren et al. [134] recently showed that the mechanism by which C. pneumoniae activates platelets is different from that of collagen and thrombin, involving activation of 12-lipoxygenase rather than COX (cyclo-oxygenase), and this interaction may not be inhibited by the COX antagonist aspirin. Accordingly, 12-lipoxygenase inhibitors may be rational therapies to reduce thrombotic risk in atherosclerosis patients with persistent C. pneumoniae infection [134].

Regarding vaccine development, the Vaccine Development and Field Trials Workshop Group [125] recommended that there is a need for: (i) improved and standardized tests for acute and chronic C. pneumoniae infection; (ii) studies to identify protective antigen delivery methods in humans; (iii) further study of the natural history and immunology of chlamydial infection to determine what constitutes a protective immune response to C. pneumoniae in humans; and (iv) epidemiology studies to characterize target groups for vaccination.

CONCLUSIONS

For the past 20 years, numerous studies have evaluated the role and importance of C. pneumoniae in atherosclerosis, but it is a major challenge to either prove or disprove a causal role for a common agent in a highly prevalent disease.

Seroepidemiological studies indicate an association between infection and cardiovascular events, and histopathological evidence confirms that C. pneumoniae is capable of persistently infecting atherosclerotic plaques in humans. The strongest evidence that C. pneumoniae is sufficient to initiate atherosclerosis and contributes to progression and plaque instability comes from experiments in rabbits and mice. Trials of antimicrobial therapy have overall been disappointing, but, in view of important limitations in study design and execution, these results cannot rule out an important pathogenic role.

Considering the totality of present evidence, C. pneumoniae is neither alone sufficient nor is it necessary to cause atherosclerosis or its clinical consequences in humans. However, C. pneumoniae is highly likely to be a modifiable risk factor that may be amenable to future therapies focused on either eradication (antibiotic therapy or early immunization) or modifying the vascular inflammatory response to infection. Future clinical studies will be necessary to define the importance of this risk across a range of populations.

Abbreviations

     
  • ACS

    acute coronary syndrome

  •  
  • ApoE

    apolipoprotein E

  •  
  • CAD

    coronary artery disease

  •  
  • EC

    endothelial cell

  •  
  • cHsp60

    chlamydial heat-shock protein 60

  •  
  • COX

    cyclo-oxygenase

  •  
  • EB

    elementary body

  •  
  • HDL

    high-density lipoprotein

  •  
  • ICAM-1

    intercellular adhesion molecule-1

  •  
  • IFN-γ

    interferon γ

  •  
  • IL

    interleukin

  •  
  • LDL

    low-density lipoprotein

  •  
  • LDLr

    LDL receptor

  •  
  • LPS

    lipopolysaccharide

  •  
  • MI

    myocardial infarction

  •  
  • AMI

    acute MI

  •  
  • MIC

    lowest antibiotic concentration at which no inclusions were seen

  •  
  • MIF

    microimmunofluorescence

  •  
  • MMP

    matrix metalloproteinase

  •  
  • MOMP

    major outer membrane protein

  •  
  • OR

    odds ratio

  •  
  • oxLDL

    oxidized LDL

  •  
  • PAD

    peripheral artery disease

  •  
  • PAI-1

    plasminogen activator inhibitor-1

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PVD

    peripheral vascular disease

  •  
  • RB

    reticulate body

  •  
  • ROXIS

    Roxithromycin in Ischemic Syndromes

  •  
  • SMC

    smooth muscle cell

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • VCAM-1

    vascular cell adhesion molecule-1

We acknowledge funding from The Wellcome Trust and the British Heart Foundation.

References

References
1
Breslow
 
J. L.
 
Cardiovascular disease burden increases, NIH funding decreases
Nat. Med.
1997
, vol. 
3
 (pg. 
600
-
601
)
2
Ross
 
R.
 
Atherosclerosis: an inflammatory disease
N. Engl. J. Med.
1999
, vol. 
340
 (pg. 
115
-
126
)
3
Yusuf
 
S.
Hawken
 
S.
Ounpuu
 
S.
, et al 
Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study
Lancet
2004
, vol. 
364
 (pg. 
937
-
952
)
4
Schaffner
 
T.
Taylor
 
K.
Bartucci
 
E. J.
, et al 
Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages
Am. J. Pathol.
1980
, vol. 
100
 (pg. 
57
-
80
)
5
Davies
 
M. J.
 
Coronary disease: the pathophysiology of acute coronary syndromes
Heart
2000
, vol. 
83
 (pg. 
361
-
366
)
6
Braunwald
 
E.
 
Unstable angina: an etiologic approach to management
Circulation
1998
, vol. 
98
 (pg. 
2219
-
2222
)
7
Glagov
 
S.
Weisenberg
 
E.
Zarins
 
C.
Stankunavicius
 
R.
Kolettis
 
G.
 
Compensatory enlargement of human atherosclerotic coronary arteries
N. Engl. J. Med.
1987
, vol. 
316
 (pg. 
1371
-
1375
)
8
Osler
 
W.
 
Olser
 
W.
MacCrae
 
T.
 
Diseases of the arteries. Modern Medicine: Its Theory and Practice in Original Contributions by Americans and Foreign Authors
1908
Philadelphia
Lea & Fabiger
(pg. 
426
-
427
)
9
Beatty
 
W. L.
Morrison
 
R. P.
Byrne
 
G. I.
 
Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis
Microbiol. Rev.
1994
, vol. 
58
 (pg. 
686
-
699
)
10
Grayston
 
J. T.
 
Background and current knowledge of Chlamydia pneumoniae and atherosclerosis
J. Infect. Dis.
2000
, vol. 
181
 (pg. 
S402
-
S410
)
11
Kuo
 
C. C.
Jackson
 
L. A.
Campbell
 
L. A.
Grayston
 
J. T.
 
Chlamydia pneumoniae (TWAR)
Clin. Microbiol. Rev.
1995
, vol. 
8
 (pg. 
451
-
461
)
12
Leinonen
 
M.
Saikku
 
P.
 
Evidence for infectious agents in cardiovascular disease and atherosclerosis
Lancet Infect. Dis.
2002
, vol. 
2
 (pg. 
11
-
17
)
13
Kuo
 
C. C.
Chen
 
H. H.
Wang
 
S. P.
Grayston
 
J. T.
 
Identification of a new group of Chlamydia psittaci strains called TWAR
J. Clin. Microbiol.
1986
, vol. 
24
 (pg. 
1034
-
1037
)
14
Yang
 
Z. P.
Kuo
 
C. C.
Grayston
 
J. T.
 
Systemic dissemination of Chlamydia pneumoniae following intranasal inoculation in mice
J. Infect. Dis.
1995
, vol. 
171
 (pg. 
736
-
738
)
15
Krull
 
M.
Klucken
 
A. C.
Wuppermann
 
F. N.
, et al 
Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae
J. Immunol.
1999
, vol. 
162
 (pg. 
4834
-
4841
)
16
Molestina
 
R. E.
Miller
 
R. D.
Ramirez
 
J. A.
Summersgill
 
J. T.
 
Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrophils and monocytes
Infect. Immun.
1999
, vol. 
67
 (pg. 
1323
-
1330
)
17
Kalayoglu
 
M. V.
Byrne
 
G. I.
 
Induction of macrophage foam cell formation by Chlamydia pneumoniae
J. Infect. Dis.
1998
, vol. 
177
 (pg. 
725
-
729
)
18
Kalayoglu
 
M. V.
Hoerneman
 
B.
LaVerda
 
D.
Morrison
 
S. G.
Morrison
 
R. P.
Byrne
 
G. I.
 
Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae
J. Infect. Dis.
1999
, vol. 
180
 (pg. 
780
-
790
)
19
Coombes
 
B. K.
Mahony
 
J. B.
 
Chlamydia pneumoniae infection of human endothelial cells induces proliferation of smooth muscle cells via an endothelial cell-derived soluble factor(s)
Infect. Immun.
1999
, vol. 
67
 (pg. 
2909
-
2915
)
20
Coombes
 
B. K.
Mahony
 
J. B.
 
cDNA array analysis of altered gene expression in human endothelial cells in response to Chlamydia pneumoniae infection
Infect. Immun.
2001
, vol. 
69
 (pg. 
1420
-
1427
)
21
Chatterjee
 
S.
Ghosh
 
N.
 
Oxidized low density lipoprotein stimulates aortic smooth muscle cell proliferation
Glycobiology
1996
, vol. 
6
 (pg. 
303
-
311
)
22
Kol
 
A.
Sukhova
 
G. K.
Lichtman
 
A. H.
Libby
 
P.
 
Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression
Circulation
1998
, vol. 
98
 (pg. 
300
-
307
)
23
Galis
 
Z. S.
Sukhova
 
G. K.
Lark
 
M. W.
Libby
 
P.
 
Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques
J. Clin. Invest.
1994
, vol. 
94
 (pg. 
2493
-
2503
)
24
Fryer
 
R. H.
Schwobe
 
E. P.
Woods
 
M. L.
Rodgers
 
G. M.
 
Chlamydia species infect human vascular endothelial cells and induce procoagulant activity
J. Investig. Med.
1997
, vol. 
45
 (pg. 
168
-
174
)
25
O'Connor
 
S.
 
Fulfillment of Koch's postulates and the causes of atherosclerosis
Am Heart J.
1999
, vol. 
138
 (pg. 
S550
-
S551
)
26
Yarnell
 
J. W.
Baker
 
I. A.
Sweetnam
 
P. M.
, et al 
Fibrinogen, viscosity, and white blood cell count are major risk factors for ischemic heart disease. The Caerphilly and Speedwell collaborative heart disease studies
Circulation
1991
, vol. 
83
 (pg. 
836
-
844
)
27
Meade
 
T. W.
Mellows
 
S.
Brozovic
 
M.
, et al 
Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study
Lancet
1986
, vol. 
ii
 (pg. 
533
-
537
)
28
Meade
 
T. W.
Vickers
 
M. V.
Thompson
 
S. G.
Seghatchian
 
M. J.
 
The effect of physiological levels of fibrinogen on platelet aggregation
Thromb. Res.
1985
, vol. 
38
 (pg. 
527
-
534
)
29
Chooi
 
C. C.
Gallus
 
A. S.
 
Acute phase reaction, fibrinogen level and thrombus size
Thromb. Res.
1989
, vol. 
53
 (pg. 
493
-
501
)
30
Becker
 
R. C.
Cannon
 
C. P.
Bovill
 
E. G.
, et al 
Prognostic value of plasma fibrinogen concentration in patients with unstable angina and non-Q-wave myocardial infarction (TIMI IIIB Trial)
Am. J. Cardiol.
1996
, vol. 
78
 (pg. 
142
-
147
)
31
Patel
 
P.
Mendall
 
M. A.
Carrington
 
D.
, et al 
Association of Helicobacter pylori and Chlamydia pneumoniae infections with coronary heart disease and cardiovascular risk factors
Br. Med. J.
1995
, vol. 
311
 (pg. 
711
-
714
)
32
Fernandez-Miranda
 
C.
Paz
 
M.
Aranda
 
J. L.
Fuertes
 
A.
Gomez De La Camara
 
A.
 
Chronic Chlamydia pneumoniae infection in patients with coronary disease. Relation with increased fibrinogen values
Med. Clin. (Barc.)
2002
, vol. 
119
 (pg. 
561
-
564
)
33
Toss
 
H.
Gnarpe
 
J.
Gnarpe
 
H.
Siegbahn
 
A.
Lindahl
 
B.
Wallentin
 
L.
 
Increased fibrinogen levels are associated with persistent Chlamydia pneumoniae infection in unstable coronary artery disease
Eur. Heart J.
1998
, vol. 
19
 (pg. 
570
-
577
)
34
Berger
 
M.
Schroder
 
B.
Daeschlein
 
G.
, et al 
Chlamydia pneumoniae DNA in non-coronary atherosclerotic plaques and circulating leukocytes
J. Lab. Clin. Med.
2000
, vol. 
136
 (pg. 
194
-
200
)
35
Leinonen
 
M.
Kerttula
 
Y.
Weber
 
T.
Saikku
 
P.
 
Acute phase response in Chlamydia pneumoniae pneumonia, Program and Abstracts of the 5th European Congress on Clinical Microbiology and Infectious Diseases
1991
 
Oslo, abstract 86
36
Laurila
 
A.
Bloigu
 
A.
Nayha
 
S.
Hassi
 
J.
Leinonen
 
M.
Saikku
 
P.
 
Chronic Chlamydia pneumoniae infection is associated with a serum lipid profile known to be a risk factor for atherosclerosis
Arterioscler. Thromb. Vasc. Biol.
1997
, vol. 
17
 (pg. 
2910
-
2913
)
37
Kaukoranta-Tolvanen
 
S. S.
Teppo
 
A. M.
Laitinen
 
K.
Saikku
 
P.
Linnavuori
 
K.
Leinonen
 
M.
 
Growth of Chlamydia pneumoniae in cultured human peripheral blood mononuclear cells and induction of a cytokine response
Microb. Pathog.
1996
, vol. 
21
 (pg. 
215
-
221
)
38
Heinemann
 
M.
Susa
 
M.
Simnacher
 
U.
Marre
 
R.
Essig
 
A.
 
Growth of Chlamydia pneumoniae induces cytokine production and expression of CD14 in a human monocytic cell line
Infect. Immun.
1996
, vol. 
64
 (pg. 
4872
-
4875
)
39
Feingold
 
K. R.
Grunfeld
 
C.
 
Tumor necrosis factor-α stimulates hepatic lipogenesis in the rat in vivo
J. Clin. Invest.
1987
, vol. 
80
 (pg. 
184
-
190
)
40
Feingold
 
K. R.
Serio
 
M. K.
Adi
 
S.
Moser
 
A. H.
Grunfeld
 
C.
 
Tumor necrosis factor stimulates hepatic lipid synthesis and secretion
Endocrinology
1989
, vol. 
124
 (pg. 
2336
-
2342
)
41
Fried
 
S. K.
Zechner
 
R.
 
Cachectin/tumor necrosis factor decreases human adipose tissue lipoprotein lipase mRNA levels, synthesis, and activity
J. Lipid Res.
1989
, vol. 
30
 (pg. 
1917
-
1923
)
42
Feingold
 
K. R.
Soued
 
M.
Serio
 
M. K.
Moser
 
A. H.
Dinarello
 
C. A.
Grunfeld
 
C.
 
Multiple cytokines stimulate hepatic lipid synthesis in vivo
Endocrinology
1989
, vol. 
125
 (pg. 
267
-
274
)
43
Feingold
 
K. R.
Grunfeld
 
C.
 
Role of cytokines in inducing hyperlipidemia
Diabetes
1992
, vol. 
41
 
Suppl. 2
(pg. 
97
-
101
)
44
Greenberg
 
A. S.
Nordan
 
R. P.
McIntosh
 
J.
Calvo
 
J. C.
Scow
 
R. O.
Jablons
 
D.
 
Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia
Cancer Res.
1992
, vol. 
52
 (pg. 
4113
-
4116
)
45
Ludewig
 
B.
Jaggi
 
M.
Dumrese
 
T.
, et al 
Hypercholesterolemia exacerbates virus-induced immunopathologic liver disease via suppression of antiviral cytotoxic T cell responses
J. Immunol.
2001
, vol. 
166
 (pg. 
3369
-
3376
)
46
Rottenberg
 
M. E.
Gigliotti Rothfuchs
 
A. C.
Gigliotti
 
D.
Svanholm
 
C.
Bandholtz
 
L.
Wigzell
 
H.
 
Role of innate and adaptive immunity in the outcome of primary infection with Chlamydia pneumoniae, as analyzed in genetically modified mice
J. Immunol.
1999
, vol. 
162
 (pg. 
2829
-
2836
)
47
Karvonen
 
M.
Tuomilehto
 
J.
Pitkaniemi
 
J.
Naukkarinen
 
A.
Saikku
 
P.
 
Importance of smoking for Chlamydia pneumoniae seropositivity
Int. J. Epidemiol.
1994
, vol. 
23
 (pg. 
1315
-
1321
)
48
von Hertzen
 
L.
Kaprio
 
J.
Koskenvuo
 
M.
Isoaho
 
R.
Saikku
 
P.
 
Humoral immune response to Chlamydia pneumoniae in twin discordant for smoking
J. Intern. Med.
1998
, vol. 
244
 (pg. 
227
-
234
)
49
Smieja
 
M.
Mahony
 
J.
Petrich
 
A.
Boman
 
J.
Chernesky
 
M.
 
Association of circulating Chlamydia pneumoniae DNA with cardiovascular disease: a systematic review
BMC Infect. Dis.
2002
, vol. 
2
 pg. 
21
 
50
von Hertzen
 
L.
Surcel
 
H. M.
Kaprio
 
J.
, et al 
Immune responses to Chlamydia pneumoniae in twins in relation to gender and smoking
J. Med. Microbiol.
1998
, vol. 
47
 (pg. 
441
-
446
)
51
Hahn
 
D. L.
Golubjatnikov
 
R.
 
Smoking is a potential confounder of the Chlamydia pneumoniae-coronary artery disease association
Arterioscler. Thromb.
1992
, vol. 
12
 (pg. 
945
-
947
)
52
Reference deleted
53
Grayston
 
J. T.
 
Infections caused by Chlamydia pneumoniae strain TWAR
Clin. Infect. Dis.
1992
, vol. 
15
 (pg. 
757
-
761
)
54
Thomas
 
L.
 
Thomas
 
L.
 
Iron (Fe)
Clinical Laboratory Diagnostics
1998
Frankfurt
Th-Books
(pg. 
273
-
277
)
55
Yuan
 
X. M.
Li
 
W.
 
The iron hypothesis of atherosclerosis and its clinical impact
Ann. Med.
2003
, vol. 
35
 (pg. 
578
-
591
)
56
Freidank
 
H. M.
Billing
 
H.
Wiedmann-Al-Ahmad
 
M.
 
Influence of iron restriction on Chlamydia pneumoniae and C. trachomatis
J. Med. Microbiol.
2001
, vol. 
50
 (pg. 
223
-
227
)
57
Kartikasari
 
A. E.
Georgiou
 
N. A.
de Geest
 
M.
, et al 
Iron enhances endothelial cell activation in response to cytomegalovirus or Chlamydia pneumoniae infection
Eur. J. Clin. Invest.
2006
, vol. 
36
 (pg. 
743
-
752
)
58
Jacobs
 
A. K.
 
Women, ischemic heart disease, revascularization, and the gender gap: what are we missing?
J. Am. Coll. Cardiol.
2006
, vol. 
47
 (pg. 
S63
-
S65
)
59
Saikku
 
P.
Leinonen
 
M.
Mattila
 
K.
, et al 
Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction
Lancet
1988
, vol. 
ii
 (pg. 
983
-
986
)
60
Bloemenkamp
 
D. G. M.
Mali
 
W. P. T. M.
Visseren
 
F. L. J.
van der Graaf
 
Y.
 
Meta-analysis of sero-epidemiologic studies of the relation between Chlamydia pneumoniae and atherosclerosis: does study design influence results?
Am. Heart J.
2003
, vol. 
145
 (pg. 
409
-
417
)
61
Danesh
 
J.
Whincup
 
P.
Walker
 
M.
, et al 
Chlamydia pneumoniae IgG titres and coronary heart disease: Prospective study and meta-analysis
Br. Med. J.
2000
, vol. 
321
 (pg. 
208
-
213
)
62
Danesh
 
J.
Whincup
 
P.
Lewington
 
S.
, et al 
Chlamydia pneumoniae IgA titres and coronary heart disease. Prospective study and meta-analysis
Eur. Heart J.
2002
, vol. 
23
 (pg. 
371
-
375
)
63
Volanen
 
I.
Jarvisalo
 
M. J.
Vainionpaa
 
R.
, et al 
Increased aortic intima-media thickness in 11-year-old healthy children with persistent Chlamydia pneumoniae seropositivity
Arterioscler. Thromb. Vasc. Biol.
2006
, vol. 
26
 (pg. 
649
-
655
)
64
Hoymans
 
V. Y.
Bosmans
 
J. M.
Van Renterghem
 
L.
, et al 
Importance of methodology in determination of Chlamydia pneumoniae seropositivity in healthy subjects and in patients with coronary atherosclerosis
J. Clin. Microbiol.
2003
, vol. 
41
 (pg. 
4049
-
4053
)
65
Maraha
 
B.
den Heijer
 
M.
Kluytmans
 
J.
Peeters
 
M.
 
Impact of serological methodology on assessment of the link between Chlamydia pneumoniae and vascular diseases
Clin. Diagn. Lab. Immunol.
2004
, vol. 
11
 (pg. 
789
-
791
)
66
Schumacher
 
A.
Lerkerod
 
A. B.
Seljeflot
 
I.
, et al 
Chlamydia pneumoniae serology: importance of methodology in patients with coronary heart disease and healthy individuals
J. Clin. Microbiol.
2001
, vol. 
39
 (pg. 
1859
-
1864
)
67
Dowell
 
S. F.
Peeling
 
R. W.
Boman
 
J.
, et al 
Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada)
Clin. Infect. Dis.
2001
, vol. 
33
 (pg. 
492
-
503
)
68
Peeling
 
R. W.
Wang
 
S. P.
Grayston
 
J. T.
, et al 
Chlamydia pneumoniae serology: interlaboratory variation in microimmunofluorescence assay results
J. Infect. Dis.
2000
, vol. 
181
 
Suppl. 3
(pg. 
S426
-
S429
)
69
Saikku
 
P.
Leinonen
 
M.
Tenkanen
 
L.
, et al 
Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study
Ann. Intern. Med.
1992
, vol. 
116
 (pg. 
273
-
278
)
70
Huittinen
 
T.
Leinonen
 
M.
Tenkanen
 
L.
, et al 
Synergistic effect of persistent Chlamydia pneumoniae infection, autoimmunity, and inflammation on coronary risk
Circulation
2003
, vol. 
107
 (pg. 
2566
-
2570
)
71
Boman
 
J.
Soderberg
 
S.
Forsberg
 
J.
, et al 
High prevalence of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in patients with cardiovascular disease and in middle-aged blood donors
J. Infect. Dis.
1998
, vol. 
178
 (pg. 
274
-
277
)
72
Aso
 
N.
Tamura
 
A.
Kadota
 
J.
Nasu
 
M.
 
Association of peripheral mononuclear cells containing Chlamydia pneumoniae DNA with acute coronary syndrome and stable coronary artery disease in Japanese subjects
Clin. Infect. Dis.
2004
, vol. 
39
 (pg. 
366
-
372
)
73
Kaul
 
R.
Uphoff
 
J.
Wiedeman
 
J.
Yadlapalli
 
S.
Wenman
 
W. M.
 
Detection of Chlamydia pneumoniae DNA in CD3+ lymphocytes from healthy blood donors and patients with coronary artery disease
Circulation
2000
, vol. 
102
 (pg. 
2341
-
2346
)
74
Airenne
 
S.
Surcel
 
H. M.
Alakarppa
 
H.
, et al 
Chlamydia pneumoniae infection in human monocytes
Infect. Immun.
1999
, vol. 
67
 (pg. 
1445
-
1449
)
75
Apfalter
 
P.
Hammerschlag
 
M. R.
Boman
 
J.
Wojta
 
J.
Huber
 
K.
Huk
 
I.
 
Reliability of nested PCR for the detection of Chlamydia pneumoniae in carotid artery atherosclerosis
Stroke
2003
, vol. 
34
 (pg. 
e73
-
e75
)
76
Shor
 
A.
Kuo
 
C. C.
Patton
 
D. L.
 
Detection of Chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques
S. Afr. Med. J.
1992
, vol. 
82
 (pg. 
158
-
161
)
77
Weiss
 
S. M.
Roblin
 
P. M.
Gaydos
 
C. A.
, et al 
Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy
J. Infect. Dis.
1996
, vol. 
173
 (pg. 
957
-
962
)
78
Ong
 
G.
Coyle
 
P.
Barros D'sa
 
A.
, et al 
Non-detection of Chlamydia species in carotid atheroma using generic primers by nested PCR in a population with a high prevalence of Chlamydia pneumoniae antibody
BMC Infect. Dis.
2001
, vol. 
1
 pg. 
12
 
79
Jantos
 
C. A.
Nesseler
 
A.
Waas
 
W.
Baumgartner
 
W.
Tillmanns
 
H.
Haberbosch
 
W.
 
Low prevalence of Chlamydia pneumoniae in atherectomy specimens from patients with coronary heart disease
Clin. Infect. Dis.
1999
, vol. 
28
 (pg. 
988
-
992
)
80
Paterson
 
D. L.
Hall
 
J.
Rasmussen
 
S. J.
Timms
 
P.
 
Failure to detect Chlamydia pneumoniae in atherosclerotic plaques of Australian patients
Pathology
1998
, vol. 
30
 (pg. 
169
-
172
)
81
Kalayoglu
 
M. V.
Libby
 
P.
Byrne
 
G. I.
 
Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease
JAMA, J. Am. Med. Assoc.
2002
, vol. 
288
 (pg. 
2724
-
2731
)
82
Sessa
 
R.
Di Pietro
 
M.
Schiavoni
 
G.
, et al 
Chlamydia pneumoniae DNA in patients with symptomatic carotid atherosclerotic disease
J. Vasc. Surg.
2003
, vol. 
37
 (pg. 
1027
-
1031
)
83
Shor
 
A.
Phillips
 
J. I.
Ong
 
G.
Thomas
 
B. J.
Taylor-Robinson
 
D.
 
Chlamydia pneumoniae in atheroma: consideration of criteria for causality
J. Clin. Pathol.
1998
, vol. 
51
 (pg. 
812
-
817
)
84
Muhlestein
 
J. B.
Hammond
 
E. H.
Carlquist
 
J. F.
, et al 
Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease
J. Am. Coll. Cardiol.
1996
, vol. 
27
 (pg. 
1555
-
1561
)
85
Fong
 
I. W.
Chiu
 
B.
Viira
 
E.
, et al 
Rabbit model for Chlamydia pneumoniae infection
J. Clin. Microbiol.
1997
, vol. 
35
 (pg. 
48
-
52
)
86
Blessing
 
E.
Lin
 
T.-M.
Campbell
 
L. A.
Rosenfeld
 
M. E.
Lloyd
 
D.
Kuo
 
C.-c.
 
Chlamydia pneumoniae induces inflammatory changes in the heart and aorta of normocholesterolemic C57BL/6J mice
Infect. Immun.
2000
, vol. 
68
 (pg. 
4765
-
4768
)
87
Blessing
 
E.
Campbell
 
L. A.
Rosenfeld
 
M. E.
Chough
 
N.
Kuo
 
C.-C.
 
Chlamydia pneumoniae infection accelerates hyperlipidemia induced atherosclerotic lesion development in C57BL/6J mice
Atherosclerosis
2001
, vol. 
158
 (pg. 
13
-
17
)
88
Blessing
 
E.
Campbell
 
L. A.
Rosenfeld
 
M. E.
Kuo
 
C.-c.
 
Chlamydia pneumoniae and hyperlipidemia are co-risk factors for atherosclerosis: infection prior to induction of hyperlipidemia does not accelerate development of atherosclerotic lesions in C57BL/6J mice
Infect. Immun.
2002
, vol. 
70
 (pg. 
5332
-
5334
)
89
Moazed
 
T. C.
Kuo
 
C.
Grayston
 
J. T.
Campbell
 
L. A.
 
Murine models of Chlamydia pneumoniae infection and atherosclerosis
J. Infect. Dis.
1997
, vol. 
175
 (pg. 
883
-
890
)
90
Aalto-Setala
 
K.
Laitinen
 
K.
Erkkila
 
L.
, et al 
Chlamydia pneumoniae does not increase atherosclerosis in the aortic root of apolipoprotein E-deficient mice
Arterioscler. Thromb. Vasc. Biol.
2001
, vol. 
21
 (pg. 
578
-
584
)
91
Caligiuri
 
G.
Rottenberg
 
M.
Nicoletti
 
A.
Wigzell
 
H.
Hansson
 
G. K.
 
Chlamydia pneumoniae infection does not induce or modify atherosclerosis in mice
Circulation
2001
, vol. 
103
 (pg. 
2834
-
2838
)
92
Ezzahiri
 
R.
Stassen
 
F. R. M.
Kurvers
 
H. A. J. M.
van Pul
 
M. M. L.
Kitslaar
 
P. J. E. H. M.
Bruggeman
 
C. A.
 
Chlamydia pneumoniae infection induces an unstable atherosclerotic plaque phenotype in LDL-receptor, ApoE double knockout mice
Eur. J. Vasc. Endovasc. Surg.
2003
, vol. 
26
 (pg. 
88
-
95
)
93
Fong
 
I. W.
Chiu
 
B.
Viira
 
E.
Jang
 
D.
Mahony
 
J. B.
 
De novo induction of atherosclerosis by Chlamydia pneumoniae in a rabbit model
Infect. Immun.
1999
, vol. 
67
 (pg. 
6048
-
6055
)
94
Hu
 
H.
Pierce
 
G. N.
Zhong
 
G.
 
The atherogenic effects of chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae
J. Clin. Invest.
1999
, vol. 
103
 (pg. 
747
-
753
)
95
Meier
 
C. R.
Derby
 
L. E.
Jick
 
S. S.
Vasilakis
 
C.
Jick
 
H.
 
Antibiotics and risk of subsequent first-time acute myocardial infarction
JAMA, J. Am. Med. Assoc.
1999
, vol. 
281
 (pg. 
427
-
431
)
96
Jackson
 
L. A.
Smith
 
N. L.
Heckbert
 
S. R.
Grayston
 
J. T.
Siscovick
 
D. S.
Psaty
 
B. M.
 
Lack of association between first myocardial infarction and past use of erythromycin, tetracycline, or doxycycline
Emerg. Infect. Dis.
1999
, vol. 
5
 (pg. 
281
-
284
)
97
Gupta
 
S.
Leatham
 
E. W.
Carrington
 
D.
Mendall
 
M. A.
Kaski
 
J. C.
Camm
 
A. J.
 
Elevated Chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction
Circulation
1997
, vol. 
96
 (pg. 
404
-
407
)
98
Wells
 
B. J.
Mainous
 
A. G.
Dickerson
 
L. M.
 
Antibiotics for the secondary prevention of ischemic heart disease: a meta-analysis of randomized controlled trials
Arch. Intern. Med.
2004
, vol. 
164
 (pg. 
2156
-
2161
)
99
Gurfinkel
 
E.
Bozovich
 
G.
Daroca
 
A.
Beck
 
E.
Mautner
 
B.
 
Randomised trial of roxithromycin in non-Q-wave coronary syndromes: ROXIS pilot study
Lancet
1997
, vol. 
350
 (pg. 
404
-
407
)
100
Gurfinkel
 
E.
Bozovich
 
G.
Beck
 
E.
, et al 
Treatment with the antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes. The final report of the ROXIS study
Eur. Heart J.
1999
, vol. 
20
 (pg. 
121
-
127
)
101
Muhlestein
 
J. B.
Anderson
 
J. L.
Carlquist
 
J. F.
, et al 
Randomized secondary prevention trial of azithromycin in patients with coronary artery disease: primary clinical results of the ACADEMIC study
Circulation
2000
, vol. 
102
 (pg. 
1755
-
1760
)
102
O'Connor
 
C. M.
Dunne
 
M. W.
Pfeffer
 
M. A.
, et al 
Azithromycin for the secondary prevention of coronary heart disease events: The WIZARD study: a randomized controlled trial
JAMA, J. Am. Med. Assoc.
2003
, vol. 
290
 (pg. 
1459
-
1466
)
103
Grayston
 
J. T.
Kronmal
 
R. A.
Jackson
 
L. A.
, et al 
Azithromycin for the secondary prevention of coronary events
N. Engl. J. Med.
2005
, vol. 
352
 (pg. 
1637
-
1645
)
104
Jespersen
 
C. M.
Als-Nielsen
 
B.
Damgaard
 
M.
, et al 
Randomised placebo controlled multicentre trial to assess short term clarithromycin for patients with stable coronary heart disease: CLARICOR trial
Br. Med. J.
2006
, vol. 
332
 (pg. 
22
-
27
)
105
Zahn
 
R.
Schneider
 
S.
Frilling
 
B.
, et al 
Antibiotic therapy after acute myocardial infarction: a prospective randomized study
Circulation
2003
, vol. 
107
 (pg. 
1253
-
1259
)
106
Cannon
 
C. P.
Braunwald
 
E.
McCabe
 
C. H.
, et al 
Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome
N. Engl. J. Med.
2005
, vol. 
352
 (pg. 
1646
-
1654
)
107
Andraws
 
R.
Berger
 
J. S.
Brown
 
D. L.
 
Effects of antibiotic therapy on outcomes of patients with coronary artery disease: a meta-analysis of randomized controlled trials
JAMA, J. Am. Med. Assoc.
2005
, vol. 
293
 (pg. 
2641
-
2647
)
108
Grayston
 
J. T.
 
Antibiotic treatment of atherosclerotic cardiovascular disease
Circulation
2003
, vol. 
107
 (pg. 
1228
-
1230
)
109
Stamm
 
W. E.
 
Potential for antimicrobial resistance in Chlamydia pneumoniae
J. Infect. Dis.
2000
, vol. 
181
 
Suppl. 3
S456
S459
110
Jones
 
R. B.
Van der Pol
 
B.
Martin
 
D. H.
Shepard
 
M. K.
 
Partial characterization of Chlamydia trachomatis isolates resistant to multiple antibiotics
J. Infect. Dis.
1990
, vol. 
162
 (pg. 
1309
-
1315
)
111
Lefevre
 
J. C.
Lepargneur
 
J. P.
 
Comparative in vitro susceptibility of a tetracycline-resistant Chlamydia trachomatis strain isolated in Toulouse (France)
Sex. Transm. Dis.
1998
, vol. 
25
 (pg. 
350
-
352
)
112
Somani
 
J.
Bhullar
 
V. B.
Workowski
 
K. A.
Farshy
 
C. E.
Black
 
C. M.
 
Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure
J. Infect. Dis.
2000
, vol. 
181
 (pg. 
1421
-
1427
)
113
Roblin
 
P. M.
Hammerschlag
 
M. R.
 
Microbiologic efficacy of azithromycin and susceptibilities to azithromycin of isolates of Chlamydia pneumoniae from adults and children with community-acquired pneumonia
Antimicrob. Agents Chemother.
1998
, vol. 
42
 (pg. 
194
-
196
)
114
Gieffers
 
J.
Rupp
 
J.
Gebert
 
A.
Solbach
 
W.
Klinger
 
M.
 
First-choice antibiotics at subinhibitory concentrations induce persistence of Chlamydia pneumoniae
Antimicrob. Agents Chemother.
2004
, vol. 
48
 (pg. 
1402
-
1405
)
115
Fong
 
I. W.
Chiu
 
B.
Viira
 
E.
, et al 
Can an antibiotic (Macrolide) prevent Chlamydia pneumoniae-induced atherosclerosis in a rabbit model?
Clin. Diagn. Lab. Immunol.
1999
, vol. 
6
 (pg. 
891
-
894
)
116
Rothstein
 
N. M.
Quinn
 
T. C.
Madico
 
G.
Gaydos
 
C. A.
Lowenstein
 
C. J.
 
Effect of azithromycin on murine arteriosclerosis exacerbated by Chlamydia pneumoniae
J. Infect. Dis.
2001
, vol. 
183
 (pg. 
232
-
238
)
117
Kalman
 
S.
Mitchell
 
W.
Marathe
 
R.
, et al 
Comparative genomes of Chlamydia pneumoniae and C. trachomatis
Nat. Genet.
1999
, vol. 
21
 (pg. 
385
-
389
)
118
Capo
 
S.
Nuti
 
S.
Scarselli
 
M.
, et al 
Chlamydia pneumoniae genome sequence analysis and identification of HLA-A2-restricted CD8+ T cell epitopes recognized by infection-primed T cells
Vaccine
2005
, vol. 
23
 (pg. 
5028
-
5037
)
119
Penttila
 
J. M.
Anttila
 
M.
Varkila
 
K.
, et al 
Depletion of CD8+ cells abolishes memory in acquired immunity against Chlamydia pneumoniae in BALB/c mice
Immunology
1999
, vol. 
97
 (pg. 
490
-
496
)
120
Chu
 
R. S.
Targoni
 
O. S.
Krieg
 
A. M.
Lehmann
 
P. V.
Harding
 
C. V.
 
CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity
J. Exp. Med.
1997
, vol. 
186
 (pg. 
1623
-
1631
)
121
Penttila
 
T.
Vuola
 
J. M.
Puurula
 
V.
, et al 
Immunity to Chlamydia pneumoniae induced by vaccination with DNA vectors expressing a cytoplasmic protein (Hsp60) or outer membrane proteins (MOMP and Omp2)
Vaccine
2000
, vol. 
19
 (pg. 
1256
-
1265
)
122
Murdin
 
A. D.
Dunn
 
P.
Sodoyer
 
R.
, et al 
Use of a mouse lung challenge model to identify antigens protective against Chlamydia pneumoniae lung infection
J. Infect. Dis.
2000
, vol. 
181
 
Suppl. 3
(pg. 
S544
-
S551
)
123
Svanholm
 
C.
Bandholtz
 
L.
Castanos-Velez
 
E.
Wigzell
 
H.
Rottenberg
 
M. E.
 
Protective DNA immunization against Chlamydia pneumoniae
Scand. J. Immunol.
2000
, vol. 
51
 (pg. 
345
-
353
)
124
Thorpe
 
C.
Edwards
 
L.
Snelgrove
 
R.
, et al 
Discovery of a vaccine antigen that protects mice from Chlamydia pneumoniae infection
Vaccine
2007
, vol. 
25
 (pg. 
2252
-
2260
)
125
Murdin
 
A. D.
Gellin
 
B.
Brunham
 
R. C.
, et al 
Collaborative multidisciplinary workshop report: progress toward a Chlamydia pneumoniae vaccine
J. Infect. Dis.
2000
, vol. 
181
 
Suppl. 3
(pg. 
S552
-
S557
)
126
Loeffler
 
F.
 
Untersuchungen über die Bedeutung der Mikroorganismen für die Entstehung der Diptherie beim Menschen, bei der Taube und beim Kalbe
1884
Gesundheitsampte
Mitth. a.d. kaiserl
(pg. 
421
-
499
)
127
Hill
 
A. B.
 
The environment and disease: association or causation?
Proc. R. Soc. Med.
1965
, vol. 
58
 (pg. 
295
-
300
)
128
Maggi
 
P.
Maserati
 
R.
Antonelli
 
G.
 
Atherosclerosis in HIV patients: a new face for an old disease?
AIDS Rev.
2006
, vol. 
8
 (pg. 
204
-
209
)
129
Bukrinsky
 
M.
Sviridov
 
D.
 
Human immunodeficiency virus infection and macrophage cholesterol metabolism
J. Leukocyte Biol.
2006
, vol. 
80
 (pg. 
1044
-
1051
)
130
Gieffers
 
J.
Durling
 
L.
Ouellette
 
S. P.
, et al 
Genotypic differences in the Chlamydia pneumoniae tyrP locus related to vascular tropism and pathogenicity
J. Infect. Dis.
2003
, vol. 
188
 (pg. 
1085
-
1093
)
131
Fujii
 
K.
Saito
 
H.
Tomioka
 
H.
Mae
 
T.
Hosoe
 
K.
 
Mechanism of action of antimycobacterial activity of the new benzoxazinorifamycin KRM-1648
Antimicrob. Agents Chemother.
1995
, vol. 
39
 (pg. 
1489
-
1492
)
132
Roblin
 
P. M.
Reznik
 
T.
Kutlin
 
A.
Hammerschlag
 
M. R.
 
In vitro activities of rifamycin derivatives ABI-1648 (Rifalazil, KRM-1648), ABI-1657, and ABI-1131 against Chlamydia trachomatis and recent clinical isolates of Chlamydia pneumoniae
Antimicrob. Agents Chemother.
2003
, vol. 
47
 (pg. 
1135
-
1136
)
132a
Jaff
 
M. R.
Constant
 
J.
Campbell
 
L. A.
, et al 
Prospective evaluation of rifalazil effect on vascular symptom of intermittent claudication (IC) and other endpoints in Chlamydia seropositive patients (The PROVIDENCE-1 trial)
Circulation
2007
, vol. 
116
 pg. 
2632
 
133
Kalvegren
 
H.
Majeed
 
M.
Bengtsson
 
T.
 
Chlamydia pneumoniae binds to platelets and triggers P-selectin expression and aggregation: a causal role in cardiovascular disease?
Arterioscler. Thromb. Vasc. Biol.
2003
, vol. 
23
 (pg. 
1677
-
1683
)
134
Kalvegren
 
H.
Andersson
 
J.
Grenegard
 
M.
Bengtsson
 
T.
 
Platelet activation triggered by Chlamydia pneumoniae is antagonized by 12-lipoxygenase inhibitors but not cyclooxygenase inhibitors
Eur. J. Pharmacol.
2007
, vol. 
566
 (pg. 
20
-
27
)
135
Mendall
 
M. A.
Carrington
 
D.
Strachan
 
D.
, et al 
Chlamydia pneumoniae: risk factors for seropositivity and association with coronary heart disease
J. Infect.
1995
, vol. 
30
 (pg. 
121
-
128
)
136
Markus
 
H. S.
Sitzer
 
M.
Carrington
 
D.
Mendall
 
M. A.
Steinmetz
 
H.
 
Chlamydia pneumoniae infection and early asymptomatic carotid atherosclerosis
Circulation
1999
, vol. 
100
 (pg. 
832
-
837
)
137
Blasi
 
F.
Cosentini
 
R.
Raccanelli
 
R.
, et al 
A possible association of Chlamydia pneumoniae infection and acute myocardial infarction in patients younger than 65 years of age
Chest
1997
, vol. 
112
 (pg. 
309
-
312
)
138
Elkind
 
M. S. V.
Tondella
 
M. L. C.
Feikin
 
D. R.
Fields
 
B. S.
Homma
 
S.
Di Tullio
 
M. R.
 
Seropositivity to Chlamydia pneumoniae is associated with risk of first ischemic stroke
Stroke
2006
, vol. 
37
 (pg. 
790
-
795
)
139
Siscovick
 
D. S.
Schwartz
 
S. M.
Corey
 
L.
, et al 
Chlamydia pneumoniae, herpes simplex virus type 1, and cytomegalovirus and incident myocardial infarction and coronary heart disease death in older adults: The Cardiovascular Health Study
Circulation
2000
, vol. 
102
 (pg. 
2335
-
2340
)
140
Arcari
 
C. M.
Gaydos
 
C. A.
Nieto
 
F. J.
Krauss
 
M.
Nelson
 
K. E.
 
Association between Chlamydia pneumoniae and acute myocardial infarction in young men in the United States military: the importance of timing of exposure measurement
Clin. Infect. Dis.
2005
, vol. 
40
 (pg. 
1123
-
1130
)
141
Mayr
 
M.
Kiechl
 
S.
Willeit
 
J.
Wick
 
G.
Xu
 
Q.
 
Infections, immunity, and atherosclerosis: associations of antibodies to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus with immune reactions to heat-shock protein 60 and carotid or femoral atherosclerosis
Circulation
2000
, vol. 
102
 (pg. 
833
-
839
)
142
Westerhout
 
C. M.
Gnarpe
 
J.
Chang
 
W. C.
, et al 
No prognostic significance of chronic infection with Chlamydia pneumoniae in acute coronary syndromes: insights from the Global Utilization of Strategies to Open Occluded Arteries IV Acute Coronary Syndromes trial
Am. Heart J.
2007
, vol. 
154
 (pg. 
306
-
312
)
143
Ridker
 
P. M.
Kundsin
 
R. B.
Stampfer
 
M. J.
Poulin
 
S.
Hennekens
 
C. H.
 
Prospective study of Chlamydia pneumoniae IgG seropositivity and risks of future myocardial infarction
Circulation
1999
, vol. 
99
 (pg. 
1161
-
1164
)
144
Wong
 
Y.-k.
Dawkins
 
K. D.
Ward
 
M. E.
 
Circulating Chlamydia pneumoniae DNA as a predictor of coronary artery disease
J. Am. Coll. Cardiol.
1999
, vol. 
34
 (pg. 
1435
-
1439
)
145
Sessa
 
R.
Di Pietro
 
M.
Schiavoni
 
G.
, et al 
Prevalence of Chlamydia pneumoniae in peripheral blood mononuclear cells in Italian patients with acute ischaemic heart disease
Atherosclerosis
2001
, vol. 
159
 (pg. 
521
-
525
)
146
Maraha
 
B.
den Heijer
 
M.
Wullink
 
M.
, et al 
Detection of Chlamydia pneumoniae DNA in buffycoat samples of patients with abdominal aortic aneurysm
Eur. J. Clin. Microbiol. Infect. Dis.
2001
, vol. 
20
 (pg. 
111
-
116
)
147
Smieja
 
M.
Chong
 
S.
Natarajan
 
M.
Petrich
 
A.
Rainen
 
L.
Mahony
 
J. B.
 
Circulating nucleic acids of Chlamydia pneumoniae and cytomegalovirus in patients undergoing coronary angiography
J. Clin. Microbiol.
2001
, vol. 
39
 (pg. 
596
-
600
)
148
Tsirpanlis
 
G.
Chatzipanagiotou
 
S.
Ioannidis
 
A.
Moutafis
 
S.
Poulopoulou
 
C.
Nicolaou
 
C.
 
Detection of Chlamydia pneumoniae in peripheral blood mononuclear cells: correlation with inflammation and atherosclerosis in haemodialysis patients
Nephrol. Dial. Transplant.
2003
, vol. 
18
 (pg. 
918
-
923
)
149
Apfalter
 
P.
Barousch
 
W.
Nehr
 
M.
Willinger
 
B.
Rotter
 
M.
Hirschl
 
A. M.
 
No evidence of involvement of Chlamydia pneumoniae in severe cerebrovascular atherosclerosis by means of quantitative real-time polymerase chain reaction
Stroke
2004
, vol. 
35
 (pg. 
2024
-
2028
)
150
Podsiadly
 
E.
Przyluski
 
J.
Kwiatkowski
 
A.
, et al 
Presence of Chlamydia pneumoniae in patients with and without atherosclerosis
Eur. J. Clin. Microbiol. Infect. Dis.
2005
, vol. 
24
 (pg. 
507
-
513
)
151
Wang
 
S. S.
Tondella
 
M. L.
Bajpai
 
A.
, et al 
Circulating Chlamydia pneumoniae DNA and advanced coronary artery disease
Int. J. Cardiol.
2007
, vol. 
118
 (pg. 
215
-
219
)
152
Blasi
 
F.
Boman
 
J.
Esposito
 
G.
, et al 
Chlamydia pneumoniae DNA detection in peripheral blood mononuclear cells is predictive of vascular infection
J. Infect. Dis.
1999
, vol. 
180
 (pg. 
2074
-
2076
)
153
Maass
 
M.
Jahn
 
J.
Gieffers
 
J.
Dalhoff
 
K.
Katus
 
H. A.
Solbach
 
W.
 
Detection of Chlamydia pneumoniae within peripheral blood monocytes of patients with unstable angina or myocardial infarction
J. Infect. Dis.
2000
, vol. 
181
 
Suppl. 3
(pg. 
S449
-
S451
)
154
Cochrane
 
M.
Pospischil
 
A.
Walker
 
P.
Gibbs
 
H.
Timms
 
P.
 
Discordant detection of Chlamydia pneumoniae in patients with carotid artery disease using polymerase chain reaction, immunofluorescence microscopy and serological methods
Pathology
2005
, vol. 
37
 (pg. 
69
-
75
)
155
Sessa
 
R.
Di Pietro
 
M.
Schiavoni
 
G.
, et al 
Measurement of Chlamydia pneumoniae bacterial load in peripheral blood mononuclear cells may be helpful to assess the state of chlamydial infection in patients with carotid atherosclerotic disease
Atherosclerosis
2007
, vol. 
195
 (pg. 
e224
-
e230
)
156
Prager
 
M.
Turel
 
Z.
Speidl
 
W. S.
, et al 
Chlamydia pneumoniae in carotid artery atherosclerosis: a comparison of its presence in atherosclerotic plaque, healthy vessels, and circulating leukocytes from the same individuals
Stroke
2002
, vol. 
33
 (pg. 
2756
-
2761
)
157
Chiu
 
B.
Viira
 
E.
Tucker
 
W.
Fong
 
I. W.
 
Chlamydia pneumoniae, cytomegalovirus, and herpes simplex virus in atherosclerosis of the carotid artery
Circulation
1997
, vol. 
96
 (pg. 
2144
-
2148
)
158
Ramirez
 
J. A.
 
Isolation of Chlamydia pneumoniae from the coronary artery of a patient with coronary atherosclerosis
Ann. Intern. Med.
1996
, vol. 
125
 (pg. 
979
-
982
)
159
Ouchi
 
K.
Fujii
 
B.
Kanamoto
 
Y.
Karita
 
M.
Shirai
 
M.
Nakazawa
 
T.
 
Chlamydia pneumoniae in coronary and iliac arteries of Japanese patients with atherosclerotic cardiovascular diseases
J. Med. Microbiol.
1998
, vol. 
47
 (pg. 
907
-
913
)
160
Grayston
 
J. T.
Kuo
 
C.-c.
Coulson
 
A. S.
, et al 
Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery
Circulation
1995
, vol. 
92
 (pg. 
3397
-
3400
)
161
Kuo
 
C. C.
Gown
 
A. M.
Benditt
 
E. P.
Grayston
 
J. T.
 
Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain
Arterioscler. Thromb.
1993
, vol. 
13
 (pg. 
1501
-
1504
)
162
Kuo
 
C. C.
Coulson
 
A. S.
Campbell
 
L. A.
, et al 
Detection of Chlamydia pneumoniae in atherosclerotic plaques in the walls of arteries of lower extremities from patients undergoing bypass operation for arterial obstruction
J. Vasc. Surg.
1997
, vol. 
26
 (pg. 
29
-
31
)
163
Kuo
 
C. C.
Grayston
 
J. T.
Campbell
 
L. A.
Goo
 
Y. A.
Wissler
 
R. W.
Benditt
 
E. P.
 
Chlamydia pneumoniae (TWAR) in coronary arteries of young adults (15–34 years old)
Proc. Natl. Acad. Sci. U.S.A.
1995
, vol. 
92
 (pg. 
6911
-
6914
)
164
Shi
 
Y.
Tokunaga
 
O.
 
Chlamydia pneumoniae and multiple infections in the aorta contribute to atherosclerosis
Pathol. Int.
2002
, vol. 
52
 (pg. 
755
-
763
)
165
Petersen
 
E.
Boman
 
J.
Persson
 
K.
, et al 
Chlamydia pneumoniae in human abdominal aortic aneurysms
Eur. J. Vasc. Endovasc. Surg.
1998
, vol. 
15
 (pg. 
138
-
142
)
166
Farsak
 
B.
Yildirir
 
A.
Akyon
 
Y.
, et al 
Detection of Chlamydia pneumoniae and Helicobacter pylori DNA in human atherosclerotic plaques by PCR
J. Clin. Microbiol.
2000
, vol. 
38
 (pg. 
4408
-
4411
)
167
Jackson
 
L. A.
Campbell
 
L. A.
Kuo
 
C. C.
Rodriguez
 
D. I.
Lee
 
A.
Grayston
 
J. T.
 
Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen
J. Infect. Dis.
1997
, vol. 
176
 (pg. 
292
-
295
)
168
Yamashita
 
K.
Ouchi
 
K.
Shirai
 
M.
Gondo
 
T.
Nakazawa
 
T.
Ito
 
H.
 
Distribution of Chlamydia pneumoniae infection in the atherosclerotic carotid artery
Stroke
1998
, vol. 
29
 (pg. 
773
-
778
)
169
Campbell
 
L. A.
O'Brien
 
E. R.
Cappuccio
 
A. L.
, et al 
Detection of Chlamydia pneumoniae TWAR in human coronary atherectomy tissues
J. Infect. Dis.
1995
, vol. 
172
 (pg. 
585
-
588
)
170
Blasi
 
F.
Denti
 
F.
Erba
 
M.
, et al 
Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms
J. Clin. Microbiol.
1996
, vol. 
34
 (pg. 
2766
-
2769
)
171
Davidson
 
M.
Kuo
 
C.-C.
Middaugh
 
J. P.
, et al 
Confirmed previous infection with Chlamydia pneumoniae (TWAR) and its presence in early coronary atherosclerosis
Circulation
1998
, vol. 
98
 (pg. 
628
-
633
)
172
Johnston
 
S. C.
Messina
 
L. M.
Browner
 
W. S.
Lawton
 
M. T.
Morris
 
C.
Dean
 
D.
 
C-reactive protein levels and viable Chlamydia pneumoniae in carotid artery atherosclerosis
Stroke
2001
, vol. 
32
 (pg. 
2748
-
2752
)
173
Gibbs
 
R. G.
Carey
 
N.
Davies
 
A. H.
 
Chlamydia pneumoniae and vascular disease
Br. J. Surg.
1998
, vol. 
85
 (pg. 
1191
-
1197
)
174
Muhlestein
 
J. B.
Anderson
 
J. L.
Hammond
 
E. H.
, et al 
Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model
Circulation
1998
, vol. 
97
 (pg. 
633
-
636
)
175
Burian
 
K.
Berencsi
 
K.
Endresz
 
V.
, et al 
Chlamydia pneumoniae exacerbates aortic inflammatory foci caused by murine cytomegalovirus infection in normocholesterolemic mice
Clin. Diagn. Lab. Immunol.
2001
, vol. 
8
 (pg. 
1263
-
1266
)
176
Burnett
 
M. S.
Gaydos
 
C. A.
Madico
 
G. E.
, et al 
Atherosclerosis in apoE knockout mice infected with multiple pathogens
J. Infect. Dis.
2001
, vol. 
183
 (pg. 
226
-
231
)
177
Moazed
 
T. C.
Campbell
 
L. A.
Rosenfeld
 
M. E.
Grayston
 
J. T.
Kuo
 
C. C.
 
Chlamydia pneumoniae infection accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice
J. Infect. Dis.
1999
, vol. 
180
 (pg. 
238
-
241
)
178
Liu
 
L.
Hu
 
H.
Ji
 
H.
Murdin
 
A. D.
Pierce
 
G. N.
Zhong
 
G.
 
Chlamydia pneumoniae infection significantly exacerbates aortic atherosclerosis in an LDLR−/− mouse model within six months
Mol. Cell. Biochem.
2000
, vol. 
215
 (pg. 
123
-
128
)
179
Liuba
 
P.
Karnani
 
P.
Pesonen
 
E.
, et al 
Endothelial dysfunction after repeated Chlamydia pneumoniae infection in apolipoprotein E-knockout mice
Circulation
2000
, vol. 
102
 (pg. 
1039
-
1044
)
180
Liuba
 
P.
Pesonen
 
E.
Paakkari
 
I.
, et al 
Co-infection with Chlamydia pneumoniae and Helicobacter pylori results in vascular endothelial dysfunction and enhanced VCAM-1 expression in apoE-knockout mice
J. Vasc. Res.
2003
, vol. 
40
 (pg. 
115
-
122
)
181
Sinisalo
 
J.
Mattila
 
K.
Valtonen
 
V.
, et al 
Effect of 3 months of antimicrobial treatment with clarithromycin in acute non-Q-wave coronary syndrome
Circulation
2002
, vol. 
105
 (pg. 
1555
-
1560
)
182
Stone
 
A. F. M.
Mendall
 
M. A.
Kaski
 
J.-C.
, et al 
Effect of treatment for Chlamydia pneumoniae and Helicobacter pylori on markers of inflammation and cardiac events in patients with acute coronary syndromes: South Thames Trial of Antibiotics in Myocardial Infarction and Unstable Angina (STAMINA)
Circulation
2002
, vol. 
106
 (pg. 
1219
-
1223
)
183
Brassard
 
P.
Bourgault
 
C.
Brophy
 
J.
, et al 
Antibiotics in primary prevention of myocardial infarction among elderly patients with hypertension
Am. Heart J.
2003
, vol. 
145
 pg. 
918
 
184
Kol
 
A.
Bourcier
 
T.
Lichtman
 
A. H.
Libby
 
P.
 
Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages
J. Clin. Invest.
1999
, vol. 
103
 (pg. 
571
-
577
)
185
Bennett
 
M. R.
 
Reactive oxygen species and death: oxidative DNA damage in atherosclerosis
Circ. Res.
2001
, vol. 
88
 (pg. 
648
-
650
)