Antibodies to Chlamydia pneumoniae are Associated with Increased Intima Media Thickness in Asymptomatic Indian Individuals

Antibodies to Chlamydia pneumoniae are Associated with Increased Intima Media Thickness in Asymptomatic Indian Individuals V. C. S. Srinivasarao Bandaru, MSc, PhD,* Subhash Kaul, MD, DM, FRCP, FAAN,* Vemu Laxmi, MD,† M. Neeraja, MSc,† M. Uma Mahesh, MD, DNB,‡ Suvarna Alladi, DM,* and Demudu Babu Boddu, MD, DM*

Chlamydia pneumoniae has been found to be associated with cerebrovascular and cardiovascular diseases in seroepidemiologic studies. The aim of this study was to investigate whether this organism is associated with increased intima media thickness (IMT) of carotid arteries in asymptomatic individuals. Serum titer of antibodies to C pneumoniae antibodies IgA and IgG in 100 asymptomatic individuals older than 40 years was measured by microimmunofluorescence. These subjects also had their IMT measured by B-mode ultrasound in the common carotid artery on both sides. Comparison of baseline characteristics between the group with abnormal IMT (.0.08 cm) and group having normal IMT (#0.08 cm) showed significant association of C pneumoniae antibodies and hypertension with the former (i.e., abnormal IMT group). Multiple logistic regression (stepwise method) established C pneumoniae as an independent risk factor for increased IMT. To conclude, this study demonstrated that C pneumoniae infection is associated with an increase of IMT in the common carotid artery. Key Words: Chlamydia pneumoniae—asymptomatic individuals—intima media thickness—erythrocyte sedimentation rate—C-reactive protein—microimmunoflourescence. Ó 2009 by National Stroke Association

Many studies published in the last decade have incriminated chronic infection as a causative factor for myocardial infarction and stroke.1,2 The support for the role of Chlamydia pneumoniae in causing the vascular events comes from the fact that many elementary bodies related to C pneumoniae have been detected in atherosclerotic plaques and fatty streaks in the aorta, coronary arteries of autopsy cases, coronary arterectomy specimens, and carotid endarterectomy specimens.3,4 The exact mechaFrom the *Departments of Neurology, †Microbiology; and ‡Radiology, Nizam’s Institute of Medical Sciences Panjagutta, Hyderabad India. Received May 18, 2008; revision received September 17, 2008; accepted September 30, 2008. Supported by a grant from Indian Council of Medical Research, New Delhi. Address correspondence to Subhash Kaul, MD, DM, FRCP, FAAN, Nizam’s Institute of Medical Sciences, Panjagutta, Hyderabad, India 500082. E-mail: [email protected]. 1052-3057/$—see front matter Ó 2009 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2008.09.020

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nism is not clear but there is evidence that infections may contribute to atherosclerotic process and may facilitate plaque rupture and thrombosis.5 A recent metaanalysis indicated that C pneumoniae may be causative for arterial disease, although further research is needed.5 An increased intima media thickness (IMT) is believed to be an early stage in the pathophysiology of atherosclerosis, and ultrasound measurement of IMT is increasingly being used in clinical research as a surrogate marker of early atherosclerosis.6 The aim of this study was to investigate whether elevated titers of antibodies to C pneumoniae are associated with increased IMT in asymptomatic individuals older than 40 years in a sample of the Indian population.

Methods Selection of Subjects The study was conducted in our neurology department, a referral university teaching hospital in south India. In all, 100 consecutive, asymptomatic individuals, older than 40

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Table 1. Comparison of baseline characteristics of asymptomatic individuals having abnormal and normal intima media thickness Parameters

Abnormal IMT, n 5 50 (%)

Normal IMT, n 5 50 (%)

Men Women Mean age, y (SD) Age range, y Chlamydia pneumoniae Hypertension Diabetes Smoking Alcoholic Hypercholesterolemia Hyperhomocysteinemia

36 (72) 14 (28) 60.4 (10.1) 44-82 18(36) 25(50) 12 (24) 12 (24) 11 (22) 23 (46) 14 (28)

39 (78) 11 (22) 54.7 (8.1) 41-75 5(10) 10(20) 7 (14) 14 (28) 13 (26) 15 (30) 11 (22)

OR

95% CI

*P value

.004 5 4 1.93 0.8 0.8 1.9 1.4

1.7-15 1.6-9.7 0.6-5.4 0.3-1.9 0.3-2 0.8-4.5 0.8-3.5

.004 .003 .3 .8 .8 .1 .6

Abbreviations: CI, confidence interval; IMT, intima media thickness; OR, odds ratio. *Chi-square test.

years, were enrolled into this study, provided they had no or history of stroke or transient ischemic attack in ocular or hemispheric distribution. This study was approved by the institutional ethical committee and study period was from January 2005 to January 2006. Data were collected through interviews of subjects by a trained research fellow, clinical assessment by the stroke neurologist, and a detailed medical record review. Standardized questions were adapted from the behavioral risk factor surveillance system7 by the Centers for Disease Control and Prevention regarding the following conditions: hypertension, diabetes, hypercholesterolemia, peripheral vascular disease, cigarette smoking, and cardiac conditions such as myocardial infarction and coronary artery disease. Standard techniques were used to measure blood pressure, height, weight, fasting blood specimen lipids (including total cholesterol, low-density lipoprotein, high-density lipoprotein, very low-density lipoprotein, and triglycerides), and glucose.8 Serum homocysteine estimation and electrocardiography was performed in all subjects. Hypertension was defined as per the guidelines of Joint National Committee VI to VII.9 Diabetes was diagnosed according to World Health Organization criteria.10 Hypercho-

lesterolemia was defined as per the 2001 guidelines of the National Institutes of Health.11 Hyperhomocysteinemia was defined as greater than 15 mg/100 mL of serum in those younger than 60 years, and greater than 20 mg/100 mL of serum in those older than 60 years.12,13 Smokers were defined as those reporting daily smoking.14 Alcoholics were defined as those in whom alcohol consumption was more than 50 g/day.15 When possible, data were obtained directly from subjects, by use of the standardized data collection instruments. When the subjects were unable to provide answers, proxy or close blood relation knowledgeable about the subject’s history was interviewed.

Color Doppler The ultrasonographic examination was performed by a qualified radiologist, experienced in neurosonology, and the findings were confirmed by the stroke neurologist, certified in neurosonology by the American Society of Neuroimaging. The sonographic evaluation was done Table 3. Correlations of C-reactive protein and Chlamydia pneumoniae positivity with intima media thickness

Table 2. Multiple logistic regression analysis (stepwise method), establishing Chlamydia pneumoniae as an independent risk factor for increased intima media thickness Variables

OR

95% CI

Dependent variable IMT Independent variables: age, sex, hypertension, diabetics, smoking, alcoholic, hyperhomocysteinemia, hypercholesterolemia, Final variable indicated in the model Chlamydia pneumoniae IgG 4.0 1.3-12.5 Abbreviations: CI, confidence interval; IMT, intima media thickness; OR, odds ratio.

Chlamydia pneumoniaepositive CRP positive Chlamydia pneumoniaenegative CRP positive

Abnormal IMT, n 5 50 (%)

Normal IMT, n 5 50 (%)

*P value

18 (36)

5 (10)

.004

10 (20)

3 (6)

.07

Abbreviations: CRP, C-reactive protein; IMT, intima media thickness. *Chi-square test.

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Table 4. Comparison of erythrocyte sedimentation rate between Chlamydia pneumoniae-positive and -negative subjects Chlamydia Chlamydia pneumoniae pneumoniae positive negative *P value Mean ESR (SD)

18.2 (5.0)

10.7 (3.2)

,.0001

Abbreviations: ESR, erythrocyte sedimentation rate. *Chi-square test.

on a high-resolution duplex ultrasound system (HDI 3000, Advance Technical Laboratories, USA) using a probe at scanner frequency of 7 to 10 MHz. Following the method used in previous similar studies, the examination included 2 cm of common carotid artery (CCA) and the carotid artery bulb. IMT on mid CCA was measured in all subjects. IMT greater than 0.08 cm was considered abnormal (Figs 1 and 2).16

Estimation of C pneumoniae Antibodies At the time of enrollment, a 5-mL blood sample was drawn after obtaining informed consent. The drawn blood samples were centrifuged and aliquoted into 1-mL specimens. These were frozen at –70 C until the time of analysis for IgG and IgA antibody titers to C pneumoniae with use of microimmunofluorescence.17

Identification of C pneumoniae The presence of C pneumoniae–specific IgG and IgA antibodies in plasma was determined by indirect immunofluorescence test using Euroimmun Biochip slide kit (Euroimmun Medizinische Labor Diagnostika, Lubeck, Germany). An IgG titer in serum of 1:100 and an IgA titer in serum of 1:100 was judged to be positive and interpreted as a marker of earlier or persistent C pneumoniae infection, respectively. The study was approved by the local institutional ethics committee.

Figure 1. mal IMT.

Carotid duplex image of longitudinal view of CCA showing nor-

Results There was a high degree of interobserver agreement between the radiologist performing the Doppler study and the neurosonologist checking the results. Kappa coefficient was 0.9, suggesting very good agreement. Comparison of baseline characteristics between the group with abnormal IMT (.0.08 cm) and the group having normal IMT (#0.08 cm) among 100 asymptomatic subjects older than 40 years showed significant association of C pneumoniae antibodies and hypertension with former (Table 1). Multiple logistic regression (stepwise method) established C pneumoniae as an independent risk factor for increased IMT (Table 2). Erythrocyte sedimentation rate and C-reactive proteins showed significantly higher values in patients with abnormal IMT and positive C pneumoniae antibody in comparison with patients with normal IMT and negative C pneumoniae (Tables 3 and 4).

Discussion This study has clearly shown an association between seropositivity for C pneumoniae and increased IMT of CCA. Elevated C pneumoniae antibody titers, age, and hypertension were found to be significant risk factors for an increased IMT in the CCA but no differences with

Statistical Analysis Statistical analysis was performed using software (Statistical Package for the Social Sciences, Version 14.0 for Windows, SPSS Inc., Chicago, IL). Means and SD for differences between groups were calculated. Odds ratio along with 95% confidence intervals were matched between the characteristics of individuals with abnormal and normal IMT. Multiple logistic regression was performed in stepwise method before and after adjustment for potential confounders. All tests were 2-sided and P values less than .05 were considered statistically significant.

Figure 2. Carotid duplex image of longitudinal view of CCA showing abnormal IMT in patient with Chlamydia pneumoniae positivity.

CHLAMYDIA PNEUMONIAE IN ASYMPTOMATIC CAROTID STENOSIS

regard to serum lipids, blood glucose, and smoking habits were found. After adjustment for potential confounding variables, the odds ratio associated with C pneumoniae infection was 4.0 (95% confidence interval, 1.3-12.5). Earlier studies have also found a relationship between C pneumoniae infection and increased IMT of the carotid artery and asymptomatic carotid artery atherosclerosis.6,18 There is enough evidence to show that carotid artery IMT is related to atherosclerosis. Previous studies have shown cross-sectional associations between CCA IMT and cardiovascular risk factors.19-23 Further, many studies have demonstrated that carotid artery IMT also predicts cardiovascular diseases such as myocardial infarction and stroke.24-26 The exact mechanism by which C pneumoniae causes increased IMT is not known although many hypotheses have been put forward. C pneumoniae infection in macrophages is considered to enhance the process of atherosclerosis by inducing production of cytokines and lipoproteins.27 Alveolar macrophages that phagocytose C pneumoniae in the lung are thought to be transported by the blood stream to the subendothelial region through the injured endothelium of the artery, subsequently stimulating endothelial proliferation and inflammation.28 The current study further strengthens the ‘‘inflammation’’ theory, as the erythrocyte sedimentation rate and C-reactive protein values were significantly higher in the group with abnormal IMT with positivity for C pneumoniae antibodies in comparison with the group with normal IMT with no C pneumoniae antibodies. Demonstration of C pneumoniae organisms in atherosclerotic lesions in coronary arteries, aorta, and carotid arteries obtained from autopsy and endarterectomy specimens supports this hypothesis.27,29 Recent evidence suggests that C pneumoniae-mediated endothelial cytotoxicity is caused by antibodies to chlamydial HSP60. Presence of serum IgA and IgG antibodies to Chlamydia infection support this hypothesis. IgA is thought to represent the chronic persistent infection and IgG, the past infection. In our study we used both IgA and IgG antibodies because they confer complementary information, because some patients do not respond to C pneumoniae infection with specific antibodies of the IgG and IgA classes. Interestingly, some studies have failed to show any relationship between C pneumoniae infection and atherosclerotic disease.30,31 The reason for this heterogeneity of response to a common risk factor across different pats of world is difficult to explain but could be a result of the phenomenon of epigenetics. In conclusion, this study demonstrated that C pneumoniae infection is associated with an increase of IMT in the CCA. The findings strengthen the evidence linking infection, atherosclerosis, and stroke.

We thank Dr. K. Venkaiah for help in statistics (Department of Statistics, National Institute of Nutrition, Hyderabad).

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