Atherosclerosis 233 (2014) 338e342
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Association between Chlamydophila pneumoniae infection and risk of coronary heart disease for Japanese: The JPHC study Naomi Sakurai-Komada a, Hiroyasu Iso b, *, Kazuko A. Koike a, c, Ai Ikeda d, Mitsumasa Umesawa a, e, Satoyo Ikehara b, Manami Inoue d, f, Shoichiro Tsugane d a
Center for Medical Sciences, School of Health Sciences, Ibaraki Prefectural University of Health Sciences, Ami, Ibaraki, Japan Public Health, Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, Suita, Osaka-fu 565-0871, Japan School of Health Sciences, Uekusagakuen University, Chiba, Japan d Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo, Japan e Department of Public Health, Dokkyo Medical University, School of Medicine, Mibu, Tochigi, Japan f Graduate School of Medicine, The University of Tokyo, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2013 Received in revised form 13 January 2014 Accepted 13 January 2014 Available online 23 January 2014
Background: Chlamydophila pneumoniae infection is considered a risk factor for atherosclerosis and coronary heart disease in western countries. However, evidence of it being a risk for Japanese is very limited because of a lower risk of coronary heart disease than for western people. The aim of this study was to examine further the association between C. pneumoniae infection and risk of coronary heart disease in Japanese. Methods: We conducted a nested caseecontrol study of 49,011 Japanese men and women who participated in The Japan Public Health Center (JPHC) study. By the end of 2004, 196 cases of coronary heart disease and 155 cases of myocardial infarction had been documented among the participants. Two controls were selected for each case. For these subjects, we examined the association between serum anti C. pneumoniae IgA and IgG on the one hand and risk of coronary heart disease on the other. Results: Concentration of C. pneumoniae IgA antibody was positively associated with risk of coronary heart disease and more specifically myocardial infarction. Subjects with the highest quartile of IgA antibody showed 2.29 (95%CI, 1.21e4.33) times higher risk of coronary heart disease and 2.58 (95%CI, 1.29e5.19) times higher risk of myocardial infarction than those with lowest quartile. However, no such association was detected for IgG antibody. Conclusion: C. pneumoniae infection was found to be positively associated with risk of coronary heart disease. Ó 2014 Published by Elsevier Ireland Ltd.
Keywords: Chlamydophila pneumoniae Coronary heart disease JPHC study Nested caseecontrol study Risk factor
Coronary heart disease has become one of the most important health problems not only in westerners but also in Asian countries. Atherosclerotic changes in coronary vessels lead to coronary heart disease, so that major risk factors of atherosclerosis, such as smoking, hypercholesterolemia, diabetes and hypertension, have also been regarded as risk factors for coronary heart disease [1,2]. In addition, acute and chronic inflammation of vessel walls has been associated with the development of atherosclerosis and subsequent coronary heart disease [3,4]. An acute phase response of bacterial infection seems to be a risk factor for coronary heart disease. For example, markers of
* Corresponding author. Tel.: þ81 6 879 3911; fax: þ81 6 879 3919. E-mail address: [email protected] (H. Iso). 0021-9150/$ e see front matter Ó 2014 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.023
inflammation as C-reactive protein and serum amyloid A were associated with risk of coronary heart disease [3,5,6]. The increment of serum amyloid A by the introduction of a lenti-virus expression vector led to the progression of atherosclerosis in apolipoprotein E-deficient mice [7]. Microbial infection also contributes to chronic inflammation. Chlamydophila pneumoniae (C. pneumoniae) has received the most attention as a candidate participant in this process [8] since electron microscopy detected C. pneumoniae in human atherosclerotic plaques, and this bacterium was isolated from human atherosclerotic coronary arteries [9,10]. C. pneumoniae infection is often asymptomatic and heals spontaneously in most cases and anti C. pneumoniae antibodies are present in over 60% of adults. However, since the antibodies do not prevent reinfection, individuals may suffer repeated C. pneumoniae
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infection throughout their life [11]. Moreover, several epidemiological studies of Westerners have demonstrated that high titers of anti C. pneumoniae are associated with risk of coronary heart disease and stroke [12e16]. For Japanese subjects, however, only limited data is available for the association between C. pneumoniae infection and risk of coronary heart disease. Two cross-sectional studies of patients with acute myocardial infarction (AMI) and controls detected higher levels of serum anti C. pneumoniae immunoglobulin G (IgG) and immunoglobulin A (IgA) in patients with acute myocardial infarction [17,18]. Moreover, our previous nested caseecontrol study showed that subjects with higher titers of IgA of C. pneumoniae were at higher risk of mortality from coronary heart disease were those with the lowest titers [19]. However, the associations between C. pneumoniae infection and risk of incident of coronary heart disease have not yet been clarified. Our a priori hypothesis was that C. pneumoniae infection leads to an increase in the risk of coronary heart disease among Japanese. We investigated this hypothesis by applying a nested caseecontrol design to a large prospective study of middle-aged Japanese men and women. 1. Methods 1.1. Study subjects The Japan Public Health Center (JPHC)-based study (cohorts I and II) was a population-based prospective study that included 140,420 subjects (68,722 men and 71,698 women). 61,595 of whom (cohort I) were 40e59 years old on December 31, 1989 (ie, born between 1930 and 1949), and 78,825 of whom (cohort II) were 40e 69 years old on December 31, 1992 (ie, born between 1923 and 1952). All participants were registered in 29 administrative districts supervised by 11 Public Health Center areas [20]. This study was approved by the human ethics review committees of the National Cancer Center and Osaka University. 1.2. Baseline survey A self-administered questionnaire was distributed to all registered, noninstitutionalized community residents between 1990 and 1995. The questionnaire included items regarding demographic characteristics, medical history, smoking and drinking habits, and diet [20]. A total of 113,461 subjects (81%; 53,375 men and 60,086 women) returned their questionnaires. The baseline health check-up was conducted from 1990 to 1992 for Cohort I and from 1993 to 1995 for Cohort II. A total of 47,910 subjects (34%; 17,246 men and 30,664 women) participated in the health checkups and 49,011 subjects (35%; 18,159 men and 30,852 women) donated a blood sample [20]. 1.3. Blood collection For blood donation, a 10 ml sample of venous blood was drawn into vacutainer tubes containing heparin. The samples were then divided into serum and buffy layers and preserved at 80 C until the analysis [20]. 1.4. Selection of cases and controls By the end of 2004, 196 cases of coronary heart disease (122 men and 74 women) had been documented among the 49,011 subjects (35%; 18,159 men and 30,852 women) who had returned their questionnaire, donated blood and had no history of myocardial infarction, angina pectoris, stroke, or cancer. These cases
339
comprised 155 cases of myocardial infarctions and 50 of sudden cardiac death within one hours. We selected two controls for each of these cases, which were matched for sex, age (within 2 years), date of blood drawn (within 3 months), time since last meal (within 4 hrs) and study location (public health center area). 1.5. Laboratory assays Blood samples were centrifuged and serum was stored at 80 C until the analysis. Serum anti C. pneumoniae immunoglobulin IgA and IgG antibodies were measured once with the aid of enzymelinked immunosorbent assay (ELISA) kits (Hitazyme C. pneumoniae antibody-IgA and -IgG kits; Hitachi Chemical, Tokyo, Japan). The sensitivity (71.7% for IgG, 73.9% for IgA) and specificity (95.8% for IgG, 92.9% for IgA) of this ELISA system had been verified beforehand by the comparing the findings obtained with the system with titers measured using a microimmunofluorescence test described by Wang and Grayston [21], a standard method for measuring anti C. pneumonia antibodies. Serum total cholesterol levels were measured with the cholesterol oxidase HDAOS method in an automatic analyzer (Hitachi 7250, Hitachi Medical Corp, Ibaraki, Japan). Serum HDL-cholesterol was measured with the modified enzymatic method (Hitachi 7600-210; Hitachi Medical Corp), and serum C-reactive protein was measured by immunonephelometric assay on a BN ProcSpec analyzer (Dade Behring, Deerfield, IL, USA). These assays were outsourced to the Koto Research Institute (Tokyo, Japan). 1.6. Confirmation of coronary heart disease and myocardial infarction Our study involved a total of 64 major hospitals in 11 public health center areas. Medical records at each hospital were reviewed by registered hospital physicians or public health center physicians who were blinded to lifestyle and laboratory data. Coronary heart disease was defined as myocardial infarction and sudden cardiac death within 1 h. Myocardial infarction was determined according to the criteria of the Monitoring Trends and Determinants of Cardiovascular Disease (MONICA) project [22], which requires data on chest pain, evidence from electrocardiograms, cardiac enzyme values, and/or autopsy. Sudden cardiac death was defined as death that occurred within 1 h from the onset of symptoms. 1.7. Statistical analysis The paired Student’s t-test and Chi-square test were used to compare the mean values of baseline cardiovascular risk factors between incident cases and controls. Crude and multivariable odds ratios (ORs) for anti C. pneumoniae IgA and IgG titers were calculated by means of logistic regression analysis. We calculated ORs for quartiles of antibody titer and commercially-defined categories. These categories for both IgA and IgG consisted of []: index between 0 and 0.89; []: index between 0.90 and 1.09; [þ]: index between 1.10 and 2.99 and [þþ]: index over 3.00. We used the lowest quartiles of antibody titer and the [] category as reference. The confounding variables in multivariable adjustment included body mass index (sex-specific quartile), alcohol intake (never, exand current drinkers), smoking status (never, ex- and current smokers), systolic blood pressure (mmHg), serum total cholesterol (mmol/L), status of diabetes mellitus (no, borderline, or yes/medication), antihypertensive medication use (yes or no) and antihyperlipidemia medication use (yes or no). We defined borderline diabetes mellitus as fasting glucose of 6.1e6.9 mmol/L or nonfasting glucose of 7.8e11.0 mmol/L, and diabetes mellitus as
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N. Sakurai-Komada et al. / Atherosclerosis 233 (2014) 338e342
fasting glucose of >7.0 mmol/L or non-fasting glucose of >11.1 mmol/L. We tested normal distribution of continuous variables before analyses and found serum CRP concentrations were skewed to the higher levels. Therefore, we converted CRP values to natural logarithm for the analyses. We used SAS version 9.3 software (SAS Institute Inc, Cary, NC) for all analyses. P-values for statistical significance were two-tailed and confidence intervals (CIs) were estimated at the 95% level. 2. Results Characteristics of the subjects are shown in Table 1. Compared with controls, cases showed higher mean values of body mass index, systolic blood pressure, serum total cholesterol and C-reactive protein, and higher proportions of diabetes mellitus, current smoking and medication use for hypertension and hyperlipidemia, but a lower proportion of current heavy drinkers. Table 2 shows crude and multivariable-adjusted ORs of coronary heart disease and myocardial infarction by quartiles of IgA and IgG titers. Even after adjustment for coronary risk factors, IgA titer was positively associated with risk of coronary heart disease and myocardial infarction. Compared with subjects with the lowest IgA titer quartile, those with the highest quartile showed 2.29 (95% confidence intervals: 1.21e4.33) and 2.58 (1.29e5.19) times higher risk of coronary heart disease and myocardial infarction, respectively. As for IgG, there was no significant association with risk of coronary heart disease or myocardial infarction. Table 3 shows crude and multivariable-adjusted ORs of coronary heart disease and myocardial infarction according to commercialbased categories of IgA and IgG titers. Compared with the subjects in category [], those in category [þ] tended to have higher risk of coronary heart disease and myocardial infarction, but this association did not reach statistical significance. 3. Discussion
Quartiles of IgA and IgG titers
Coronary heart disease IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa Myocardial infarction IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa
The main findings of our prospective nested caseecontrol study were that, according to the quartile-based analysis, C. pneumoniae antibody IgA titer analysis was positively associated with risk of coronary heart disease, more specifically myocardial infarction, but this was not the case for IgG titer. The commercial-based category, however, was not significantly associated with this risk because the [] category included subjects with higher risk of coronary heart disease. Table 1 Baseline characteristics by case and control.
Male, % Age, year Body Mass Index, kg/m2 Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Total cholesterol, mmol/L Diabetes mellitus % Current smoker, % Current drinker, % Medication use for hypertension, % Medication use for hyperlipidemia, % C-reactive protein, mg/dL
Table 2 Crude and multivariable odds ratios (ORs) and 95% confidence intervals (95% CIs) of incident of coronary heart disease and myocardial infarction according to quartiles of IgA and IgG titers
Cases (n ¼ 196) Mean (SD)/Median/ Percentage
Controls (n ¼ 392) Mean (SD)/Median/ Percentage
62.2 57.1 (7.6) 24.4 (3.2)* 141.3 (19.7)*
62.2 57.0 (7.6) 23.8 (3.0) 134.2 (17.0)
82.4 (10.8)*
79.7 (9.8)
5.48 (1.04)* 13.2* 39.5* 36.4* 33.2*
5.21 (1.04) 3.4 28.2 46.2 17.9
3.6
1.5
0.08*
0.06
*P < 0.05 (difference between cases and controls).
1 (low)
2
3
4 (high)
0e0.45 129 31 1.00
0.45e0.71 148 50 1.71 (1.00e2.90) 2.03 (1.11e3.74)
0.71e1.10 159 60 2.11 (1.21e3.68) 2.36 (1.27e4.41)
1.11e6.06 152 55 1.99 (1.13e3.49) 2.29 (1.21e4.33)
0.45e0.86 142 43 1.08 (0.65e1.81) 1.45 (0.80e2.60)
0.86e1.54 149 51 1.39 (0.78e2.45) 1.49 (0.76e2.92)
1.54e4.64 158 60 1.69 (0.94e3.02) 1.85 (0.92e3.72)
0.45e0.71 107 33 1.45 (0.82e2.56) 1.77 (0.92e3.43)
0.72e1.10 125 48 2.17 (1.20e3.94) 2.58 (1.30e5.12)
1.10e4.25 114 45 2.27 (1.24e4.15) 2.58 (1.29e5.19)
0.45e0.86 117 36 1.06 (0.61e1.84) 1.23 (0.65e2.30)
0.86e1.50 113 42 1.52 (0.82e2.84) 1.44 (0.69e3.01)
1.54e4.65 108 38 1.42 (0.74e2.73) 1.25 (0.56e2.79)
1.00
0e0.45 139 42 1.00 1.00
0e0.45 119 29 1.00 1.00
0e0.45 127 39 1.00 1.00
a Adjusted for body mass index(sex-specific quartile), ethanol intake (3 categories), smoking status (3 categories), systolic blood pressure level, serum total cholesterol level, status of diabetes mellitus (3 categories), medication use for hypertension, medication use for hypercholesterolemia.
Our result was consistent with the findings of studies of Westerners, which also found a significant association between IgA titer and risk of coronary heart disease [23], but not IgG [24]. In addition, a previous nested caseecontrol study of Japanese showed that women with the highest tertile of C. pneumoniae antibody titers of IgA showed 2.7 times higher mortality from coronary heart disease compared with those with the lowest tertile, but, again, this was not the case for IgG titer [19]. The half-life of IgA is about one week, while that of IgG is several years. Thus, a high IgA titer may reflect repeated or persistent infection, while the IgG titer may reflect past infection. Our result and previous findings suggest that repeated infection of C. pneumoniae is indeed a risk factor of coronary heart disease. Repeated infection may cause repeated acute phase response of bacterial infection. According to an animal study [7], the increment of serum amyloid A, a marker of acute phase inflammation, by the introduction of a lenti-virus expression vector was associated with the progression of atherosclerosis. In that experiment, there was a dose-dependent chemotaxis of serum amyloid A concentrations to macrophages in atherosclerotic regions [7]. C. pneumoniae spreads from the respiratory tract to the systemic vascular system through infected monocytes or macrophages via the blood or lymphatic circulation [25]. C. pneumoniae infection
N. Sakurai-Komada et al. / Atherosclerosis 233 (2014) 338e342 Table 3 Crude and multivariable odds ratios (ORs) and 95% confidence intervals (95% CIs) of incident of coronary heart disease and myocardial infarction according to commercial-based categories of IgA and IgG titers.
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infection of C. pneumoniae may lead to an increase in the risk of coronary heart disease. Acknowledgments
Commercial-based categories of IgA and IgG titers
Coronary heart disease IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa Myocardial infarction IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa
þ
þþ
0e0.89 359 115 1.00
0.90e1.10 73 26 1.19 (0.69e2.04) 1.31 (0.71e2.41)
1.10e2.97 149 53 1.18 (0.78e1.79) 1.25 (0.78e2.01)
3.01e6.06 7 2 0.86 (0.17e4.47) 1.54 (0.20e11.89)
0.90e1.09 56 19 1.28 (0.68e2.39) 1.14 (0.56e2.35)
1.10e2.94 217 79 1.45 (0.94e2.24) 1.27 (0.76e2.12)
3.06e4.64 25 10 1.79 (0.72e4.43) 1.43 (0.50e4.10)
0.90e1.10 56 21 1.40 (0.75e2.58) 1.75 (0.87e3.50)
1.10e2.69 113 44 1.49 (0.93e2.38) 1.57 (0.91e2.71)
3.01e4.25 5 1 0.58 (0.06e5.24) 0.87 (0.05e14.02)
0.90e1.09 46 16 1.27 (0.64e2.53) 1.32 (0.61e2.86)
1.10e2.90 150 54 1.39 (0.84e2.29) 1.04 (0.57e1.90)
3.05e4.64 17 7 1.73 (0.61e4.92) 1.43 (0.44e4.68)
1.00
0e0.90 290 88 1.00 1.00
0e0.89 291 89 1.00 1.00
0e0.90 252 78 1.00 1.00
a Adjusted for body mass index(sex-specific quartile), ethanol intake (3 categories), smoking status (3 categories), systolic blood pressure level, serum total cholesterol level, status of diabetes mellitus (3 categories), medication use for hypertension, medication use for hypercholesterolemia.
induces endothelial dysfunction [26], platelet aggregation via Pselectin expression [27], and the expression of procoagulant protein such as plasminogen activator inhibitor I and proinflammatory cytokine such as interleukin-6 [28]. All of these processes may contribute to the initiation and development of atherosclerosis in coronary arteries. The strengths of the present study include the study design, study subjects and endpoints. First, we used a prospective study. Prospective design allows for making careful adjustments for important potential confounding variables, such as body mass index, smoking, alcohol intake, blood pressure, serum glucose and cholesterol, and medication use. Second, our results were derived from a general population, which is more suitable for generalization than results from occupational, hospital-based, or volunteer samples. Third, the incidence of coronary heart disease is a more directly relevant than mortality for estimating risk factors because mortality is also influenced by medical treatment. The limitation of this study is that only a single blood sampling was obtained at the baseline examination. This means that we could not investigate the potential effect of C. pneumoniae infection after the baseline examination and of recurrent infection. In conclusion, our nested caseecontrol study of middle-aged Japanese men and women suggests that repeated or persistent
Sources of funding: This research was supported by a Grant-inAid for Cancer Research and by the Third-Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare, Japan and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan (Research Project Number: 19390174). The authors wish to thank all staff members in each study location where this study was conducted and in the central office for their painstaking efforts in conducting the baseline survey and follow-up. References [1] Soler EP, Ruiz VC. Epidemiology and risk factors of cerebral ischemia and ischemic heart diseases: similarities and differences. Curr Cardiol Rev 2010;6: 138e49. [2] Braunwald E. Shattuck lecture e cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med 1997;337: 1360e9. [3] Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836e43. [4] Stoll G, Bendszus M. Inflammation and atherosclerosis: novel insights into plaque formation and destabilization. Stroke 2006;37:1923e32. [5] Buckley DI, Fu R, Freeman M, Rogers K, Helfand M. C-reactive protein as a risk factor for coronary heart disease: a systematic review and meta-analyses for the U.S. Preventive Services Task Force. Ann Intern Med 2009;151:483e95. [6] Danesh J, Whincup P, Walker M, et al. Low grade inflammation and coronary heart disease: prospective study and updated meta-analyses. BMJ 2000;321: 199e204. [7] Dong Z, Wu T, Qin W, et al. Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. Mol Med 2011;17:1357e64. [8] Kalayoglu Murat V, Libby Peter, Byrne Gerald I. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA 2002;288:2724e31. [9] Jackson LA, Campbell LA, Kuo CC, Rodriguez DI, Lee A, Grayston JT. Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen. J Infect Dis 1997;176:292e5. [10] Higuchi M, Castelli JB, Aiello VD, et al. Great amount of C. pneumoniae in ruptured plaque vessel segments at autopsy. A comparative study with stable plaques. Arq Bras Cardiol 2000;74:149e51. [11] Grayston JT, Campbell LA, Kuo CC, et al. A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR. J Infect Dis 1990;161:618e25. [12] 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;116:273e8. [13] Wimmer ML, Sandmann-Strupp R, Saikku P, Haberl RL. Association of chlamydial infection with cerebrovascular disease. Stroke 1996;27:2207e10. [14] Nieto FJ, Folsom AR, Sorlie PD, Grayston JT, Wang SP, Chambless LE. Chlamydia pneumoniae infection and incident coronary heart disease: the atherosclerosis risk in communities study. Am J Epidemiol 1999;150:149e56. [15] Cook PJ, Honeybourne D, Lip GY, Beevers DG, Wise R, Davies P. Chlamydia pneumoniae antibody titers are significantly associated with acute stroke and transient cerebral ischemia: the West Birmingham Stroke Project. Stroke 1998;29:404e10. [16] Elkind MS, Lin IF, Grayston JT, Sacco RL. Chlamydia pneumoniae and the risk of first ischemic stroke: the Northern Manhattan Stroke Study. Stroke 2000;31: 1521e5. [17] Kosaka C, Hara K, Komiyama Y, Takahashi H. Possible role of chronic infection with Chlamydia pneumoniae in Japanese patients with acute myocardial infarction. Jpn Circ J 2000;64:819e24. [18] Miyashita N, Toyota E, Sawayama T, et al. Association of chronic infection of Chlamydia pneumoniae and coronary heart disease in the Japanese. Intern Med 1998;37:913e6. [19] Sakurai-Komada N, Koike KA, Kaku Y, Hiraki M, Cui R, Sankai T, et al. Chlamydia pneumoniae infection was associated with risk of mortality from coronary heart disease in Japanese women but not men: the JACC Study. J Atheroscler Thromb 2010;17:510e6. [20] Tsugane S, Sobue T. Baseline survey of JPHC study-design and participation rate. Japan Public Health Center-based prospective study on cancer and cardiovascular diseases. J Epidemiol 2001;11:S24e9. [21] Wang SP, Grayston JT. Immunologic relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. Am J Ophthalmol 1970;70:367e74. [22] Tunstall-Pedoe H, Kuulasmaa K, Amouyel P, Arveiler D, Rajakangas AM, Pajak A. Myocardial infarction and coronary deaths in the World Health
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Association between Chlamydophila pneumoniae infection and risk of coronary heart disease for Japanese: The JPHC study Naomi Sakurai-Komada a, Hiroyasu Iso b, *, Kazuko A. Koike a, c, Ai Ikeda d, Mitsumasa Umesawa a, e, Satoyo Ikehara b, Manami Inoue d, f, Shoichiro Tsugane d a
Center for Medical Sciences, School of Health Sciences, Ibaraki Prefectural University of Health Sciences, Ami, Ibaraki, Japan Public Health, Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, Suita, Osaka-fu 565-0871, Japan School of Health Sciences, Uekusagakuen University, Chiba, Japan d Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo, Japan e Department of Public Health, Dokkyo Medical University, School of Medicine, Mibu, Tochigi, Japan f Graduate School of Medicine, The University of Tokyo, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2013 Received in revised form 13 January 2014 Accepted 13 January 2014 Available online 23 January 2014
Background: Chlamydophila pneumoniae infection is considered a risk factor for atherosclerosis and coronary heart disease in western countries. However, evidence of it being a risk for Japanese is very limited because of a lower risk of coronary heart disease than for western people. The aim of this study was to examine further the association between C. pneumoniae infection and risk of coronary heart disease in Japanese. Methods: We conducted a nested caseecontrol study of 49,011 Japanese men and women who participated in The Japan Public Health Center (JPHC) study. By the end of 2004, 196 cases of coronary heart disease and 155 cases of myocardial infarction had been documented among the participants. Two controls were selected for each case. For these subjects, we examined the association between serum anti C. pneumoniae IgA and IgG on the one hand and risk of coronary heart disease on the other. Results: Concentration of C. pneumoniae IgA antibody was positively associated with risk of coronary heart disease and more specifically myocardial infarction. Subjects with the highest quartile of IgA antibody showed 2.29 (95%CI, 1.21e4.33) times higher risk of coronary heart disease and 2.58 (95%CI, 1.29e5.19) times higher risk of myocardial infarction than those with lowest quartile. However, no such association was detected for IgG antibody. Conclusion: C. pneumoniae infection was found to be positively associated with risk of coronary heart disease. Ó 2014 Published by Elsevier Ireland Ltd.
Keywords: Chlamydophila pneumoniae Coronary heart disease JPHC study Nested caseecontrol study Risk factor
Coronary heart disease has become one of the most important health problems not only in westerners but also in Asian countries. Atherosclerotic changes in coronary vessels lead to coronary heart disease, so that major risk factors of atherosclerosis, such as smoking, hypercholesterolemia, diabetes and hypertension, have also been regarded as risk factors for coronary heart disease [1,2]. In addition, acute and chronic inflammation of vessel walls has been associated with the development of atherosclerosis and subsequent coronary heart disease [3,4]. An acute phase response of bacterial infection seems to be a risk factor for coronary heart disease. For example, markers of
* Corresponding author. Tel.: þ81 6 879 3911; fax: þ81 6 879 3919. E-mail address: [email protected] (H. Iso). 0021-9150/$ e see front matter Ó 2014 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.023
inflammation as C-reactive protein and serum amyloid A were associated with risk of coronary heart disease [3,5,6]. The increment of serum amyloid A by the introduction of a lenti-virus expression vector led to the progression of atherosclerosis in apolipoprotein E-deficient mice [7]. Microbial infection also contributes to chronic inflammation. Chlamydophila pneumoniae (C. pneumoniae) has received the most attention as a candidate participant in this process [8] since electron microscopy detected C. pneumoniae in human atherosclerotic plaques, and this bacterium was isolated from human atherosclerotic coronary arteries [9,10]. C. pneumoniae infection is often asymptomatic and heals spontaneously in most cases and anti C. pneumoniae antibodies are present in over 60% of adults. However, since the antibodies do not prevent reinfection, individuals may suffer repeated C. pneumoniae
N. Sakurai-Komada et al. / Atherosclerosis 233 (2014) 338e342
infection throughout their life [11]. Moreover, several epidemiological studies of Westerners have demonstrated that high titers of anti C. pneumoniae are associated with risk of coronary heart disease and stroke [12e16]. For Japanese subjects, however, only limited data is available for the association between C. pneumoniae infection and risk of coronary heart disease. Two cross-sectional studies of patients with acute myocardial infarction (AMI) and controls detected higher levels of serum anti C. pneumoniae immunoglobulin G (IgG) and immunoglobulin A (IgA) in patients with acute myocardial infarction [17,18]. Moreover, our previous nested caseecontrol study showed that subjects with higher titers of IgA of C. pneumoniae were at higher risk of mortality from coronary heart disease were those with the lowest titers [19]. However, the associations between C. pneumoniae infection and risk of incident of coronary heart disease have not yet been clarified. Our a priori hypothesis was that C. pneumoniae infection leads to an increase in the risk of coronary heart disease among Japanese. We investigated this hypothesis by applying a nested caseecontrol design to a large prospective study of middle-aged Japanese men and women. 1. Methods 1.1. Study subjects The Japan Public Health Center (JPHC)-based study (cohorts I and II) was a population-based prospective study that included 140,420 subjects (68,722 men and 71,698 women). 61,595 of whom (cohort I) were 40e59 years old on December 31, 1989 (ie, born between 1930 and 1949), and 78,825 of whom (cohort II) were 40e 69 years old on December 31, 1992 (ie, born between 1923 and 1952). All participants were registered in 29 administrative districts supervised by 11 Public Health Center areas [20]. This study was approved by the human ethics review committees of the National Cancer Center and Osaka University. 1.2. Baseline survey A self-administered questionnaire was distributed to all registered, noninstitutionalized community residents between 1990 and 1995. The questionnaire included items regarding demographic characteristics, medical history, smoking and drinking habits, and diet [20]. A total of 113,461 subjects (81%; 53,375 men and 60,086 women) returned their questionnaires. The baseline health check-up was conducted from 1990 to 1992 for Cohort I and from 1993 to 1995 for Cohort II. A total of 47,910 subjects (34%; 17,246 men and 30,664 women) participated in the health checkups and 49,011 subjects (35%; 18,159 men and 30,852 women) donated a blood sample [20]. 1.3. Blood collection For blood donation, a 10 ml sample of venous blood was drawn into vacutainer tubes containing heparin. The samples were then divided into serum and buffy layers and preserved at 80 C until the analysis [20]. 1.4. Selection of cases and controls By the end of 2004, 196 cases of coronary heart disease (122 men and 74 women) had been documented among the 49,011 subjects (35%; 18,159 men and 30,852 women) who had returned their questionnaire, donated blood and had no history of myocardial infarction, angina pectoris, stroke, or cancer. These cases
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comprised 155 cases of myocardial infarctions and 50 of sudden cardiac death within one hours. We selected two controls for each of these cases, which were matched for sex, age (within 2 years), date of blood drawn (within 3 months), time since last meal (within 4 hrs) and study location (public health center area). 1.5. Laboratory assays Blood samples were centrifuged and serum was stored at 80 C until the analysis. Serum anti C. pneumoniae immunoglobulin IgA and IgG antibodies were measured once with the aid of enzymelinked immunosorbent assay (ELISA) kits (Hitazyme C. pneumoniae antibody-IgA and -IgG kits; Hitachi Chemical, Tokyo, Japan). The sensitivity (71.7% for IgG, 73.9% for IgA) and specificity (95.8% for IgG, 92.9% for IgA) of this ELISA system had been verified beforehand by the comparing the findings obtained with the system with titers measured using a microimmunofluorescence test described by Wang and Grayston [21], a standard method for measuring anti C. pneumonia antibodies. Serum total cholesterol levels were measured with the cholesterol oxidase HDAOS method in an automatic analyzer (Hitachi 7250, Hitachi Medical Corp, Ibaraki, Japan). Serum HDL-cholesterol was measured with the modified enzymatic method (Hitachi 7600-210; Hitachi Medical Corp), and serum C-reactive protein was measured by immunonephelometric assay on a BN ProcSpec analyzer (Dade Behring, Deerfield, IL, USA). These assays were outsourced to the Koto Research Institute (Tokyo, Japan). 1.6. Confirmation of coronary heart disease and myocardial infarction Our study involved a total of 64 major hospitals in 11 public health center areas. Medical records at each hospital were reviewed by registered hospital physicians or public health center physicians who were blinded to lifestyle and laboratory data. Coronary heart disease was defined as myocardial infarction and sudden cardiac death within 1 h. Myocardial infarction was determined according to the criteria of the Monitoring Trends and Determinants of Cardiovascular Disease (MONICA) project [22], which requires data on chest pain, evidence from electrocardiograms, cardiac enzyme values, and/or autopsy. Sudden cardiac death was defined as death that occurred within 1 h from the onset of symptoms. 1.7. Statistical analysis The paired Student’s t-test and Chi-square test were used to compare the mean values of baseline cardiovascular risk factors between incident cases and controls. Crude and multivariable odds ratios (ORs) for anti C. pneumoniae IgA and IgG titers were calculated by means of logistic regression analysis. We calculated ORs for quartiles of antibody titer and commercially-defined categories. These categories for both IgA and IgG consisted of []: index between 0 and 0.89; []: index between 0.90 and 1.09; [þ]: index between 1.10 and 2.99 and [þþ]: index over 3.00. We used the lowest quartiles of antibody titer and the [] category as reference. The confounding variables in multivariable adjustment included body mass index (sex-specific quartile), alcohol intake (never, exand current drinkers), smoking status (never, ex- and current smokers), systolic blood pressure (mmHg), serum total cholesterol (mmol/L), status of diabetes mellitus (no, borderline, or yes/medication), antihypertensive medication use (yes or no) and antihyperlipidemia medication use (yes or no). We defined borderline diabetes mellitus as fasting glucose of 6.1e6.9 mmol/L or nonfasting glucose of 7.8e11.0 mmol/L, and diabetes mellitus as
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fasting glucose of >7.0 mmol/L or non-fasting glucose of >11.1 mmol/L. We tested normal distribution of continuous variables before analyses and found serum CRP concentrations were skewed to the higher levels. Therefore, we converted CRP values to natural logarithm for the analyses. We used SAS version 9.3 software (SAS Institute Inc, Cary, NC) for all analyses. P-values for statistical significance were two-tailed and confidence intervals (CIs) were estimated at the 95% level. 2. Results Characteristics of the subjects are shown in Table 1. Compared with controls, cases showed higher mean values of body mass index, systolic blood pressure, serum total cholesterol and C-reactive protein, and higher proportions of diabetes mellitus, current smoking and medication use for hypertension and hyperlipidemia, but a lower proportion of current heavy drinkers. Table 2 shows crude and multivariable-adjusted ORs of coronary heart disease and myocardial infarction by quartiles of IgA and IgG titers. Even after adjustment for coronary risk factors, IgA titer was positively associated with risk of coronary heart disease and myocardial infarction. Compared with subjects with the lowest IgA titer quartile, those with the highest quartile showed 2.29 (95% confidence intervals: 1.21e4.33) and 2.58 (1.29e5.19) times higher risk of coronary heart disease and myocardial infarction, respectively. As for IgG, there was no significant association with risk of coronary heart disease or myocardial infarction. Table 3 shows crude and multivariable-adjusted ORs of coronary heart disease and myocardial infarction according to commercialbased categories of IgA and IgG titers. Compared with the subjects in category [], those in category [þ] tended to have higher risk of coronary heart disease and myocardial infarction, but this association did not reach statistical significance. 3. Discussion
Quartiles of IgA and IgG titers
Coronary heart disease IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa Myocardial infarction IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa
The main findings of our prospective nested caseecontrol study were that, according to the quartile-based analysis, C. pneumoniae antibody IgA titer analysis was positively associated with risk of coronary heart disease, more specifically myocardial infarction, but this was not the case for IgG titer. The commercial-based category, however, was not significantly associated with this risk because the [] category included subjects with higher risk of coronary heart disease. Table 1 Baseline characteristics by case and control.
Male, % Age, year Body Mass Index, kg/m2 Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Total cholesterol, mmol/L Diabetes mellitus % Current smoker, % Current drinker, % Medication use for hypertension, % Medication use for hyperlipidemia, % C-reactive protein, mg/dL
Table 2 Crude and multivariable odds ratios (ORs) and 95% confidence intervals (95% CIs) of incident of coronary heart disease and myocardial infarction according to quartiles of IgA and IgG titers
Cases (n ¼ 196) Mean (SD)/Median/ Percentage
Controls (n ¼ 392) Mean (SD)/Median/ Percentage
62.2 57.1 (7.6) 24.4 (3.2)* 141.3 (19.7)*
62.2 57.0 (7.6) 23.8 (3.0) 134.2 (17.0)
82.4 (10.8)*
79.7 (9.8)
5.48 (1.04)* 13.2* 39.5* 36.4* 33.2*
5.21 (1.04) 3.4 28.2 46.2 17.9
3.6
1.5
0.08*
0.06
*P < 0.05 (difference between cases and controls).
1 (low)
2
3
4 (high)
0e0.45 129 31 1.00
0.45e0.71 148 50 1.71 (1.00e2.90) 2.03 (1.11e3.74)
0.71e1.10 159 60 2.11 (1.21e3.68) 2.36 (1.27e4.41)
1.11e6.06 152 55 1.99 (1.13e3.49) 2.29 (1.21e4.33)
0.45e0.86 142 43 1.08 (0.65e1.81) 1.45 (0.80e2.60)
0.86e1.54 149 51 1.39 (0.78e2.45) 1.49 (0.76e2.92)
1.54e4.64 158 60 1.69 (0.94e3.02) 1.85 (0.92e3.72)
0.45e0.71 107 33 1.45 (0.82e2.56) 1.77 (0.92e3.43)
0.72e1.10 125 48 2.17 (1.20e3.94) 2.58 (1.30e5.12)
1.10e4.25 114 45 2.27 (1.24e4.15) 2.58 (1.29e5.19)
0.45e0.86 117 36 1.06 (0.61e1.84) 1.23 (0.65e2.30)
0.86e1.50 113 42 1.52 (0.82e2.84) 1.44 (0.69e3.01)
1.54e4.65 108 38 1.42 (0.74e2.73) 1.25 (0.56e2.79)
1.00
0e0.45 139 42 1.00 1.00
0e0.45 119 29 1.00 1.00
0e0.45 127 39 1.00 1.00
a Adjusted for body mass index(sex-specific quartile), ethanol intake (3 categories), smoking status (3 categories), systolic blood pressure level, serum total cholesterol level, status of diabetes mellitus (3 categories), medication use for hypertension, medication use for hypercholesterolemia.
Our result was consistent with the findings of studies of Westerners, which also found a significant association between IgA titer and risk of coronary heart disease [23], but not IgG [24]. In addition, a previous nested caseecontrol study of Japanese showed that women with the highest tertile of C. pneumoniae antibody titers of IgA showed 2.7 times higher mortality from coronary heart disease compared with those with the lowest tertile, but, again, this was not the case for IgG titer [19]. The half-life of IgA is about one week, while that of IgG is several years. Thus, a high IgA titer may reflect repeated or persistent infection, while the IgG titer may reflect past infection. Our result and previous findings suggest that repeated infection of C. pneumoniae is indeed a risk factor of coronary heart disease. Repeated infection may cause repeated acute phase response of bacterial infection. According to an animal study [7], the increment of serum amyloid A, a marker of acute phase inflammation, by the introduction of a lenti-virus expression vector was associated with the progression of atherosclerosis. In that experiment, there was a dose-dependent chemotaxis of serum amyloid A concentrations to macrophages in atherosclerotic regions [7]. C. pneumoniae spreads from the respiratory tract to the systemic vascular system through infected monocytes or macrophages via the blood or lymphatic circulation [25]. C. pneumoniae infection
N. Sakurai-Komada et al. / Atherosclerosis 233 (2014) 338e342 Table 3 Crude and multivariable odds ratios (ORs) and 95% confidence intervals (95% CIs) of incident of coronary heart disease and myocardial infarction according to commercial-based categories of IgA and IgG titers.
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infection of C. pneumoniae may lead to an increase in the risk of coronary heart disease. Acknowledgments
Commercial-based categories of IgA and IgG titers
Coronary heart disease IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa Myocardial infarction IgA Range of IgA titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa IgG Range of IgG titers Number of subjects Number of cases Crude OR and 95% CI Multivariable OR and 95% CIa
þ
þþ
0e0.89 359 115 1.00
0.90e1.10 73 26 1.19 (0.69e2.04) 1.31 (0.71e2.41)
1.10e2.97 149 53 1.18 (0.78e1.79) 1.25 (0.78e2.01)
3.01e6.06 7 2 0.86 (0.17e4.47) 1.54 (0.20e11.89)
0.90e1.09 56 19 1.28 (0.68e2.39) 1.14 (0.56e2.35)
1.10e2.94 217 79 1.45 (0.94e2.24) 1.27 (0.76e2.12)
3.06e4.64 25 10 1.79 (0.72e4.43) 1.43 (0.50e4.10)
0.90e1.10 56 21 1.40 (0.75e2.58) 1.75 (0.87e3.50)
1.10e2.69 113 44 1.49 (0.93e2.38) 1.57 (0.91e2.71)
3.01e4.25 5 1 0.58 (0.06e5.24) 0.87 (0.05e14.02)
0.90e1.09 46 16 1.27 (0.64e2.53) 1.32 (0.61e2.86)
1.10e2.90 150 54 1.39 (0.84e2.29) 1.04 (0.57e1.90)
3.05e4.64 17 7 1.73 (0.61e4.92) 1.43 (0.44e4.68)
1.00
0e0.90 290 88 1.00 1.00
0e0.89 291 89 1.00 1.00
0e0.90 252 78 1.00 1.00
a Adjusted for body mass index(sex-specific quartile), ethanol intake (3 categories), smoking status (3 categories), systolic blood pressure level, serum total cholesterol level, status of diabetes mellitus (3 categories), medication use for hypertension, medication use for hypercholesterolemia.
induces endothelial dysfunction [26], platelet aggregation via Pselectin expression [27], and the expression of procoagulant protein such as plasminogen activator inhibitor I and proinflammatory cytokine such as interleukin-6 [28]. All of these processes may contribute to the initiation and development of atherosclerosis in coronary arteries. The strengths of the present study include the study design, study subjects and endpoints. First, we used a prospective study. Prospective design allows for making careful adjustments for important potential confounding variables, such as body mass index, smoking, alcohol intake, blood pressure, serum glucose and cholesterol, and medication use. Second, our results were derived from a general population, which is more suitable for generalization than results from occupational, hospital-based, or volunteer samples. Third, the incidence of coronary heart disease is a more directly relevant than mortality for estimating risk factors because mortality is also influenced by medical treatment. The limitation of this study is that only a single blood sampling was obtained at the baseline examination. This means that we could not investigate the potential effect of C. pneumoniae infection after the baseline examination and of recurrent infection. In conclusion, our nested caseecontrol study of middle-aged Japanese men and women suggests that repeated or persistent
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