Chlamydial infection and progression of carotid atherosclerosis in patients on regular haemodialysis

Akihiko Kato1, Takako Takita2, Yukitaka Maruyama2 and Akira Hishida3

1 Renal, Endocrine and Metabolic Division, Shizuoka Cancer Center Hospital, Nagaizumi-cho, Shizuoka 411-8777, 2 Maruyama Hospital, Hamamatsu 430-0903 and 3 First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan

Correspondence and offprint requests to: Akihiko Kato, MD, Renal, Endocrine and Metabolic Division, Shizuoka Cancer Center Hospital, Nagaizumi-cho, Shizuoka 411-8777, Japan. Email: a.kato{at}scch.jp



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Recent findings have suggested a possible contribution of chlamydial infection to the pathogenesis of atherosclerosis in the general population. However, the role that chlamydial antibody status plays in atherosclerosis generation in haemodialysis (HD) patients remains uncertain.

Methods. We measured carotid artery intima medial thickness (IMT) over 4 years in 100 HD subjects (age: 58±10 years; time on HD: 13±7 years; male/female: 67/33) and examined potential associations between Chlamydia pneumoniae (Cp) antibody seropositivity and changes in carotid artery IMT.

Results. During 4 years, carotid artery IMT increased significantly from 0.62±0.13 to 0.73±0.12 mm (P< 0.01). IMT progression was significantly and positively correlated with age (r = 0.37, P<0.01), log-transformed C-reactive protein (CRP; r = 0.33, P<0.01) and log-transformed interleukin-6 (IL-6; r = 0.22, P<0.04), but inversely correlated with blood creatinine (r = –0.36, P<0.01) and albumin (r = –0.24, P<0.02). IMT increases were more prominent in patients positive for IgA antibodies (0.039± 0.022 mm/year, n = 52) compared with those without IgA antibodies (0.025±0.032 mm/year, n = 48) (P<0.01). IgA seropositivity did not accelerate IMT progression in patients with increased CRP (>0.11 mg/dl, n = 53), but significantly increased IMT to a greater extent in IgA-positive subjects than in IgA-negative subjects having lower CRP (≤0.11 mg/dl, n = 47) (0.017±0.024 vs 0.034± 0.021 mm/year; P = 0.01). Multivariate regression analysis revealed that serum creatinine, log-transformed CRP and IgA Cp seropositivity were independent risk factors for IMT progression (P<0.01). In contrast, IgG Cp antibody did not affect IMT progression or carotid plaque formation.

Conclusions. IMT progression is associated with inflammation and malnutrition. In addition, persistent chlamydial infection may be associated with IMT progression, but only in HD patients having low blood CRP.

Keywords: carotid wall intima medial thickness; Chlamydia pneumoniae; haemodialysis; hypoalbuminaemia; interleukin-6



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cardiovascular disease is the main cause of morbidity and mortality in subjects with end-stage renal disease (ESRD) undergoing regular haemodialysis (HD). High-resolution ultrasound measurement of intima medial thickness (IMT) at large arteries is a useful tool to assess the severity of atherosclerosis. When using this method, young ESRD adults had greater increases in carotid artery IMT compared with matched control subjects [1]. Hyperphosphataemia increased arterial wall thickness in uraemic rats [2]. These findings indicate that uraemia and/or metabolic disorders due to ESRD, rather than classical cardiovascular risk factors, are associated with carotid atherosclerosis. Advanced carotid artery IMT is also an indicator of mortality in dialysis patients [3]. For example, a 0.1 mm increase in carotid artery IMT predicted 31 and 41% higher risks of all-cause and cardiovascular 5 year mortality, respectively, in regular HD patients [3].

It was demonstrated recently that infectious burden may be involved in the atherosclerotic processes of dialysis patients. An example is the suggested association between chlamydial infection and atherosclerosis. For instance, IgG anti-Chlamydia titres were correlated with the number of carotid plaques in smokers [4]. In addition, immunoglobulin G (IgG) Chlamydia pneumoniae (Cp) seropositivity was associated with advanced IMT and coronary calcifications in young ESRD adults [1]. An association between IgA antibodies and carotid intima medial area was observed in pre-dialysis subjects [5]. IgA titres were associated with maximal diameter of carotid plaque in maintenance HD patients [6]. Stenvinkel et al. [7] also showed a higher prevalence of IgA CP seropositivity in patients having enhanced carotid intima medial area during the first year of dialysis. A higher prevalence of IgA Cp positivity was found in ESRD patients showing angiographic presence of coronary artery disease [8]. However, almost all of these studies were cross-sectional and it remains to be determined whether chronic chlamydial exposure over many years causes progression of carotid atherosclerosis in chronic dialysis patients.

The main goal of the present study was to ascertain whether chronic chlamydial status is associated with advancing atherosclerosis in uraemic subjects. We prospectively followed 100 chronic HD patients for 4 years and examined whether IgA and IgG seropositivity to Cp affects progression of carotid artery IMT and plaque formation. We also examined whether inflammatory and nutritional status influences ultrasound changes in carotid atherosclerosis in subjects receiving regular HD.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Study population
We initially measured ultrasound imaging in carotid arteries in 157 chronic HD patients from a single dialysis unit (Maruyama Hospital) and then followed the patients for the next 4 years. During this period, 15 patients died and two received kidney transplantation. Twelve out of the 15 deaths were due to cardiovascular causes. In addition, three patients had transferred to other dialysis clinics. Because of time limitations during our measurements, we failed to obtain consent in 37 patients to conduct carotid echography at the end of follow-up. Therefore, serial measurements in 100 patients (63.7%) were available for the end-point analysis. Of these, 67 were male and 33 were female with a mean age of 58±10 years (range: 33–80 years). At the start of follow-up, the mean time on HD treatment was 13±7 years (range: 3–30 years). The causes of ESRD were primary renal disease in 88 and diabetes in 12 patients. Residual urine outputs were within 200 ml/day in all patients. Thirty-eight subjects (38%) were habitual smokers and the life-long dose of smoking was calculated as pack-years of smoking, a product of years of smoking history and daily number of cigarettes. There were 33 subjects positive for anti-hepatitis C virus (HCV) antibodies. Two patients had been receiving statins during the follow-up.

HD-related factors
All patients had been given regular HD for 4–5 h at three times per week with a blood flow rate of 180–220 ml/min. All patients used bicarbonate dialysate (30 mEq/l; Kindaly AF-2P, Fuso, Osaka, Japan) at a dialysis flow rate of 500 ml/min. All HD treatments were performed using one of the following membranes: low-flux ultrafiltration rate (UFR <20 ml/mmHg/h) modified regenerated cellulose hollow-fibre (MRC; AM-SD, Asahi Medical, Tokyo, Japan or CL-EE, Terumo, Tokyo, Japan), medium-flux (UFR 20–40 ml/mmHg/h) cellulose triacetate hollow-fibre (CTA; FB-U, Nipro Medical, Osaka, Japan or TFW, Teijin-Gambro, Tokyo, Japan) or high-flux (UFR >40 ml/mmHg/h) polysulphone synthetic hollow-fibre (PS; BS-U, Toray Medical, Tokyo, Japan or APA, Asahi Medical, Tokyo, Japan). None of the patients refused a dialyser. There were no bacteria or pyrogens detected in the dialysate, which was prepared from water obtained by reverse osmosis. Samples obtained from the endotoxin removal filter and tested by routine analysis with Limulus amebocyte lysate assay (Wako Junyaku endotoxin measurement kit; Wako, Tokyo, Japan) showed that endotoxin concentrations were <20 EU/l during the observation period. Blood samples were drawn from the arterial site of the arteriovenous fistula at the start and at the end of dialysis after the 2 day interval. The efficiency of dialysis was assessed from the urea reduction rate (URR), calculated from monthly blood tests and based on the formula (1 – post-BUN/pre-BUN) x 100, and the delivered dose of dialysis (Kt/Vurea) using a single-pool urea kinetic model. Protein catabolic rate (PCR), an indirect indicator of protein intake, was determined from dialysis urea removal and serum urea levels.

Analytical procedures
At study onset, we measured serum urea nitrogen, creatinine, total protein, albumin, total cholesterol, high-density lipoprotein (HDL)-cholesterol and haemoglobin by standard laboratory techniques using an automatic analyser. Intact parathyroid hormone (PTH) was determined by immunoradiometric assay. C-reactive protein (CRP) was measured with a laser nepherometer. Since blood CRP is easily changeable, we measured CRP both at the entry and 2 months later and calculated its mean value. Serum interleukin-6 (IL-6) was determined by enzyme-linked immunosorbent assay (ELISA; Human IL-6 Ultrasensitive ELISA, Biosource International, Camarillo, CA). The coefficient of intra-assay variation for IL-6 replicates was 4.5%. Plasma total homocysteine (Hcy) was determined by high-pressure liquid chromatography based on a derivative SBD-F fluorescence.

At 2 years later, we determined IgG- and IgA-specific antibodies for Cp using an ELISA kit (Hitazyme C. pneumoniae Ab-IgG, Ab-IgA; Hitachi-Kasei, Tokyo, Japan). This test can selectively detect the antigen of chlamydial outer membrane complex of elementary body. The sensitivities of IgG and IgA antibodies were 87.5 and 72.7% and the specificities for IgG and IgA antibodies were 95.7 and 93.1%, respectively. The cut-off value (COV) that distinguished between positive and negative samples was calculated from the formula: cut-off value = (CN x K) + 0.20, where CN is the mean value of negative control serum adsorbances at 405 nm in duplicate and K is the ratio of positive serum sample value to the mean values of positive control serum adsorbances at 405 nm. Seropositivity of IgG and IgA for Cp was defined as cut-off index (COI) >1.10, where COI represents the ratio between absorbance of sample at 450 nm x K and COV. We were not able to measure Cp antibodies in 5 out of 13 dead patients, because they had expired during the first 2 years.

Evaluation of carotid atherosclerosis
Examination of plaques was performed using a 7.5 MHz linear array transducer with high-resolution B-mode echography (Aloka, Tokyo, Japan) just before HD, both at the beginning and the end of the study. The probe discriminates distances of 0.1 mm. The subjects were in the supine position with the head slightly turned away from the echography unit during the measurement. All ultrasound evaluations were conducted by the same investigator who was unaware of the laboratory characteristics of the subjects and of the results of the first measurements. These were performed at the time of inclusion and at follow-up. The wall of the common carotid artery, measured at 0.5–1.0 cm proximal to the beginning of the carotid bulb, was assessed using similar IMT methods for both measurements. The IMT was defined as the distance between the leading edge of the lumen–intima echo of the near wall and the leading edge of the media–adventitia echo. A frozen longitudinal view was captured and recorded at the position of the visually thickest part of the wall. IMT was always performed at plaque-free regions. The mean IMT values were calculated from at least two repeated measurements for each artery. The intra-observer coefficient of variation in all of the patients was 6.0% (mean difference: 0.02 mm) in our analysis [3]. The interobserver coefficient variation was 9.2% (mean difference: 0.03 mm) [3]. The common, internal and external carotid arteries were also scanned longitudinally and transversely to assess occurrence of plaques. We defined presence of carotid plaque as intima–media thickening that exceeded >1.0 mm. If a plaque was present, a frozen B-mode image of the thickest part of the plaque in the longitudinal view was recorded. We conducted a single assessment of carotid atherosclerosis at the onset and at the end of the study and evaluated the difference between the mean IMT and maximal diameter during the 4 year follow-up. The annual decrease or increase in IMT (mm/year) and the percentile change of maximal plaque diameter during 4 years were calculated for each patient. We divided all patients into quartiles of {Delta}IMT progression: group 1 had the lowest quartile of {Delta}IMT (<0.010, n = 19); group 2 had the second quartile of {Delta}IMT (0.010 ≤ {Delta}IMT< 0.030, n = 26); group 3 had the third quartile of {Delta}IMT (0.030 ≤ {Delta}IMT<0.050, n = 29); and group 4 had the largest quartile of {Delta}IMT (≥0.050, n = 26).

The pre-dialysis mean arterial pressure (MAP) was determined as MAP = diastolic + (systolic–diastolic)/3. Low-density lipoprotein (LDL)-cholesterol was calculated from a modified Friedewald's formula for the Japanese population (total cholesterol–HDL-cholesterol–triglyceride/4).

Statistical analysis
Data are expressed as means±SD. Differences between the two groups were analysed using unpaired Student's t-tests following the analysis of variance method. P-values of <0.05 were considered statistically significant. Adjusted multiple comparisons were performed using the Bonferroni method. Correlations were determined with linear regression analysis. Independent associations between one dependent variable and two independent variables were assessed with stepwise multiple regression analysis. Since IL-6 and CRP were highly skewed, these values were natural log-transformed before they were entered into the analysis. All statistical calculations were performed with StatView software (SAS Institute Inc., Cary, NC, USA).



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Changes in carotid artery imaging
Carotid artery IMT increased from 0.62±0.07 to 0.73±0.12 mm over 4 years. An annual increase in IMT ({Delta}IMT, mm/year) ranged from –0.025 to 0.106 mm/year, with a median of 0.031 mm/year. The prevalence of carotid plaque significantly increased from 30.0 to 50% during the observation (P< 0.01). There was no difference in mean maximal carotid diameters between both measurements [3.3±1.3 vs 2.9±1.2 mm; P = not significant (NS)]. There was no difference in {Delta}IMT between diabetic patients (n = 12) and non-diabetics (n = 88) (0.042±0.025 vs 0.031±0.028 mm/year; P = NS). There were also no differences in the prevalence of plaque formation or maximal plaque diameter between diabetic and non-diabetic subjects. In patients seropositive for anti-HCV antibody (n = 16), there was a significantly larger maximal diameter in carotid plaque compared with seronegative patients (n = 34) at the second measurement (3.4±1.8 vs 2.7±0.8 mm; P<0.05). However, HCV infection did not accelerate {Delta}IMT during the 4 year period (0.033±0.029 vs 0.032±0.028 mm/year; P = NS).

Association between IMT progression and clinical parameters
There was a significant and positive correlation between {Delta}IMT and age (r = 0.37, P<0.01; Figure 1). No association was found between initial IMT and {Delta}IMT during the 4 years. A significant and inverse relationship was found between {Delta}IMT and serum creatinine (r = –0.36, P<0.01) and albumin (r = –0.23, P<0.02; Figure 2). Since mean CRP (median: 0.11 mg/dl; range: 0.01–9.15 mg/dl) and IL-6 (median: 1.35 pg/ml; range: 0.39–5.36 pg/ml) were non-normally distributed, we log-transformed these parameters. A significant and positive association was also found between {Delta}IMT and log-transformed CRP (r = 0.33, P<0.01) and log-transformed IL-6 (r = 0.22, P<0.04) (Figure 3). In contrast, {Delta}IMT did not correlate with time on HD, blood Hcy, haemoglobin, LDL-cholesterol and intact PTH. There was no association between {Delta}IMT and dialysis efficacy, assessed from Kt/Vurea and URR. The percentile change in carotid plaque diameter was significantly and negatively correlated with PCR (r = –0.49, P<0.01; n = 30). In contrast, blood CRP and IL-6 were not associated with the percentile changes of maximal plaque diameters.



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Fig. 1. A positive relation between age and carotid artery IMT progression. There was a positive and significant relationship between patient age and IMT progression in HD subjects (r = 0.37, P<0.01).

 


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Fig. 2. Nutritional parameters and carotid artery IMT progression. IMT progression was significantly and inversely correlated with serum albumin (r = –0.23, P<0.02) and creatinine (r = –0.36, P<0.01).

 


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Fig. 3. Positive associations between IMT progression and log-transformed CRP and IL-6. A positive and significant relation was found between IMT and log-transformed circulating CRP (r = 0.33, P<0.01) and IL-6 (r = 0.22, P<0.04).

 
Significant differences for age and serum creatinine were found between quartile 1 for {Delta}IMT and quartiles 3 and 4 (Table 1). No differences were found in the prevalence of males, smokers or PS membrane among the four quartiles. A significantly lower seropositivity for IgA antibody to Cp was found in quartile 1 compared with the other three quartiles that increased their IMT by >0.010 mm/year (P<0.01). The prevalence of IgG seropositivity was smaller in quartile 1, but this difference did not reach statistical significance. There were no differences in other atherogenic risk factors, such as LDL-cholesterol and MAP between quartiles 1 and 4 (Table 1).


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Table 1. Characteristics of four subgroups divided by progression of carotid artery IMT

 
Chlamydial infection and carotid atherosclerosis
Seropositive rates of IgA and IgG antibodies to Cp were 52 and 64, respectively. Forty-three out of 52 (82.7%) IgA antibody-positive subjects had elevated circulating IgG antibody. Age, diabetes, male gender and anti-HCV antibody were not associated with IgA or IgG seropositivity. Habitual smokers had a significantly higher prevalence of IgG chlamydial antibody compared with non-smokers (76.3 vs 56.5%; P<0.05). In contrast, smoking did not affect IgA seropositivity (52.6 vs 52.6%; P = NS).

In Cp IgA-positive patients (n = 52), {Delta}IMT was more greatly increased than in Cp IgA-negative patients (n = 48) (P = 0.01), despite a significantly lower IMT at the beginning of the study (Table 2). In contrast, the prevalence of carotid plaque was comparable between IgA seropositive and seronegative patients during the 4 year follow-up. There were also no differences in maximal plaque diameter change, both at inclusion and at the end of follow-up between both groups. Anti-chlamydial IgG seropositivity did not increase {Delta}IMT or plaque formation (Table 3). Blood albumin was identical in seropositive and seronegative patients (3.8±0.3 vs 3.8±0.3 g/dl; P = NS).


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Table 2. Cp seropositivity and echographic findings in HD patients: anti-Cp IgA antibody

 

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Table 3. Cp seropositivity and echographic findings in HD patients: anti-Cp IgG antibody

 
We next examined whether inflammatory status alters the effect of IgA Cp seropositivity on {Delta}IMT. In patients with blood CRP lower than the median value (≤0.11 mg/dl, n = 47), there was a significant increase in {Delta}IMT in IgA Cp seropositive patients (0.034±0.021 mm/year, n = 24) compared with the seronegative subjects (0.017±0.024 mm/year, n = 23) (P = 0.01). In contrast, in patients with mean CRP greater than the median value (>0.11 mg/dl, n = 53), there was no significant difference in {Delta}IMT between seropositive (0.043±0.023 mm/year, n = 28) and seronegative (0.033±0.036 mm/year, n = 25) subjects (Figure 4). Blood CRP and IgA seropositivity did not change the prevalence of carotid plaque during the follow-up.



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Fig. 4. CRP elevation, IgA Cp seropositivity and IMT progression in non-diabetic patients. In patients with low CRP (≤0.11 mg/dl, n = 47), a significantly greater IMT increase was found in IgA Cp seropositive subjects (n = 23) than in seronegative subjects (n = 24) (P<0.01). In contrast, Cp seropositivity did not change IMT progression in subjects with higher CRP (>0.11 mg/dl, n = 53).

 
In patients negative for both anti-HCV and Cp-IgA antibodies (n = 11), {Delta}IMT was not increased during the 4 year observation (–0.001±0.009 mm/year). There was a significantly greater {Delta}IMT in patients seropositive for both antibodies (0.041±0.024 mm/year, n = 12) compared with seronegative patients (P<0.01).

Independent factors for IMT progression
To evaluate the influence of different risk factors for {Delta}IMT, a stepwise regression analysis was performed. A variety of risk factors, including age, male sex, diabetes, smoking, time on HD, serum creatinine, albumin, LDL-cholesterol, log-transformed CRP, log-transformed IL-6 and IgG and IgA Cp seropositivity, were selected as independent variables; {Delta}IMT was the dependent variable. We found that log-transformed CRP, smoking habit, patient age, IgA Cp seropositivity and blood creatinine were significant predictors of {Delta}IMT (Table 4).


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Table 4. Determinants of IMT progression

 
Cardiovascular events during follow-up
During 4 years, only six cardiovascular events had occurred in subjects (congestive heart failure, n = 2; peripheral arterial occlusive disease, n = 2; acute myocardial infarction, n = 1; angina pectoris, n = 1). There was no difference in the prevalence of cardiovascular events between IgA antibody seropositive and seronegative subjects (3.8 vs 8.3%; P = NS). HD vascular access thrombosis occurred in 38 patients, but IgA seropositivity did not increase the prevalence of these events (42.3% vs 33.3%; P = NS).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Several lines of evidence support the hypothesis that persistent chlamydial infection contributes to mortality and incidence of ischaemic heart disease in the general population. Dialysis subjects were reported to have a higher prevalence of Cp DNA in peripheral white blood cells than in healthy controls (16.3 vs 8.5%) [9]. However, the clinical relevance of Cp infection remains controversial in ESRD subjects.

Haubitz and Brunkhorst [10] first demonstrated that Cp seropositivity was associated with shorter survival and increased cardiovascular events in peritoneal dialysis patients over a 7 year observation period. A higher incidence of cardiovascular events was also reported in HD patients positive for IgA anti-Cp antibodies [11,12]. In the present study, we found that IgA Cp seropositivity significantly increased IMT progression in HD patients. However, this association was observed only in subjects with low CRP (≤0.11 mg/dl). A recent report also showed that Cp infection did not increase risk of mortality above expected levels in patients with raised CRP [13]. Thus, persistent Cp infection may accelerate atherogenic risk in uraemic subjects with low inflammatory condition.

Inflammation and malnutrition are well-known factors contributing to carotid atherosclerosis in dialysis subjects. Carotid artery IMT has been positively correlated with circulating CRP and IL-6, but inversely correlated with albumin in maintenance HD patients [3–5]. Malnourished patients had a higher intima medial area and a higher prevalence of carotid plaques compared with well-nourished patients [14]. However, these studies were cross-sectional and there have been no available studies that prospectively examined the impact of poor nutritional and inflammatory states on progression of IMT in dialysis patients. In the present study, we showed that {Delta}IMT was significantly and positively correlated with log-transformed CRP and IL-6, but significantly and negatively with serum albumin and creatinine. Multivariate regression analysis revealed that log-transformed CRP and creatinine were independent risk factors for progression of IMT. These observations support the importance of inflammation and malnutrition in the advancement of atherosclerosis in HD patients.

In mice lacking the LDL receptor, a cholesterol-enriched diet increased blood cholesterol and induced atherosclerotic lesions in the aorta [15]. Cp infection only aggravated atherosclerosis when the mice were hyperlipidaemic, indicating that Cp may accelerate the development of disease in these animals. In support of this, Cp also can initiate and propagate inflammatory processes that potentially contribute to atherosclerotic changes [16]. Thus, chlamydial infection may accelerate atherosclerosis through lipid- and inflammatory-related processes.

A recent meta-analysis showed that IgG seropositive patients had a 1.6-fold higher relative risk for cardiovascular events in the general population [17]. However, significance was lost when only prospective case-controlled studies were selected. In addition, the duration of follow-up in prospective studies was inversely related to the strength of the relation [17]. In the current study, we also found no association between IgG Cp seropositivity and IMT progression and cardiovascular accidents during 4 years of observation. These findings indicate that Cp IgG antibody titres may not provide an appropriate means to determine the status of exposure to chronic chlamydial infection.

A recent cross-sectional study showed a negative association between Cp infection and carotid artery IMT in regular HD patients [18]. The cause of the disparity between this and the present study is unclear, but may be due in part to a single measurement of the infectious pathogen. Recently, an aggregate value for pathogen burden rather than exposure to a specific pathogen has been shown to be more important for the risk of cardiovascular events in the general population [19,20]. An increasing pathogen burden was associated with a higher risk of myocardial infarction and death among non-renal patients with coronary artery disease [19]. In addition, a greater IMT progression was observed in subjects seropositive to more than four infectious pathogens than in those seropositive to less than three pathogens, regardless of pathogen type [20]. Thus, if similar conditions are present in uraemic patients, the contribution of an individual pathogen may have been masked in these studies. Further studies will be necessary to determine the relationship between total pathogen burden and progressive atherosclerosis in ESRD patients.

Hepatitis C viral infection has been demonstrated as an independent risk factor for the development of carotid plaques and IMT in the general population [21,22]. Ishizaka et al. [21] showed HCV seropositivity is associated with 1.9- and 2.9-fold risks of carotid artery plaque and IMT, respectively, in normal subjects not having severe liver dysfunction. However, there have been no studies examining HCV infection and carotid atherosclerosis in dialysis patients. In this study, we found that HCV-positive patients had a significantly greater maximal plaque diameter at the end of follow-up. We also found a greater increase in {Delta}IMT in subjects positive for both anti-HCV and Cp IgA antibodies than in subjects negative for both antibodies. Because elevations in blood HCV core protein are independently associated with carotid plaque formation [22], additional studies will be needed to ascertain its relationship to atherosclerosis in ESRD patients by using a more sensitive marker of active virus replication, such as HCV core antigen or HCV RNA.

In this study, HD subjects had an IMT change of 0.032±0.028 mm/year. During the 4 year observation period, IMT increased by an average of 0.128 mm, which is close to the discrimination limit (0.1 mm) of the 7.5 MHz echo probe used in this study. Although we did not measure IMT in non-renal failure controls in this study, the increase in HD subjects appeared to be greater than in the non-uraemic population (0.012 mm/year) [23].

There were some important limitations in the present study. First, we measured anti-chlamydial antibodies only at the middle-point during the follow-up. Since the half lives of IgA and IgG antibodies are 5–7 days and weeks to months [24], respectively, the titres that we measured may or may not have been positive in the first 2 year period of the follow-up. Similarly, we did not assess other methods, such as microimmunofluorescence assay or PCR products from peripheral cells. In HD patients, Cp seropositivity has been shown to be less reliable as a marker to assess presence of Cp [25]. Second, we only assessed inflammatory parameters at study onset. We, therefore, used the mean of two CRP measurements from 2 months to overcome this limitation, because acute-phase reactants vary with time in dialysis patients [26]. Third, we did not exclude inherent problems that influence serological testing. In HD subjects, a variant of the myeloperoxidase gene is associated with Cp seropositivity [27]. Fourth, we probably underestimated the prevalence of cardiovascular events, since we excluded from the study at least 12 patients who expired from cardiovascular events. Finally, our patients were aged, had a low frequency of diabetes and had received long-term HD. Thus, our data may not apply to younger or diabetic subjects that spent shorter times on HD.

In summary, the present study showed that annual IMT progression was associated with nutritional and inflammatory conditions. In addition, IgA seropositivity for Cp was independently associated with progression of carotid artery IMT in HD subjects. IgA Cp seropositivity increased IMT progression only in patients having CRP ≤0.11 mg/dl. In contrast, there was no relationship between the occurrence of carotid plaque and anti-chlamydial antibody seropositivity. These findings suggest that persistent chlamydial exposure may play a role in the accelerated carotid atherosclerosis found, especially in HD subjects with low CRP.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 11. 2.04
Accepted in revised form: 6. 5.04





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