There are contrasting results in studies of cardiovascular risk in patients with genotype 1 chronic hepatitis C (G1 CHC). We evaluated the prevalence of carotid atherosclerosis compared with a control population in order to assess the potential association between atherosclerosis, host and viral factors, and liver histological features. In all, 174 consecutive biopsy-proven G1 CHC patients were evaluated by anthropometric and metabolic measurements and 174 patients attending an outpatient cardiology unit were used as controls. Intima-media thickness (IMT) and carotid plaques, defined as focal thickening of >1.3 mm at the level of common carotid, were evaluated using ultrasonography. All G1 CHC biopsies were scored by one pathologist for staging and grading, and graded for steatosis. Carotid plaques were found in 73 (41.9%) G1 CHC patients compared with 40 (22.9%) control patients (P < 0.001). Similarly, G1 CHC patients had a greater IMT compared with control patients (1.04 ± 0.21 versus 0.90 ± 0.16; P < 0.001). Multivariate logistic regression analysis showed that older age (odds ratio [OR] 1.047, 95% confidence interval [CI]: 1.014-1.082, P = 0.005), and severe hepatic fibrosis (OR 2.177, 95% CI: 1.043-4.542, P = 0.03), were independently linked to the presence of carotid plaques. In patients ≤55 years, 15/67 cases with F0-F2 fibrosis (22.3%) had carotid plaques, compared with 11/21 (52.3%) with F3-F4 fibrosis (P = 0.008). By contrast, in patients >55 years the prevalence of carotid plaques was similar in those with or without severe fibrosis (25/43, 58.1% versus 22/43, 51.1%; P = 0.51). Conclusion: Severe hepatic fibrosis is associated with a high risk of early carotid atherosclerosis in G1 CHC patients. (HEPATOLOGY 2012)
Several clinical studies have shown that metabolic disorders, namely, type 2 diabetes,1 insulin resistance (IR),2 and hepatic steatosis,3 are highly prevalent in patients with genotype 1 chronic hepatitis C (G1 CHC). Experimental and clinical studies have indicated that hepatitis C virus (HCV) is able to directly induce metabolic and inflammatory alterations, and is responsible for the occurrence of both steatosis and IR.4, 5
Despite a definite link between HCV infection and metabolic abnormalities, few studies have evaluated the cardiovascular risk in HCV-infected patients compared with noninfected patients and the results are contrasting. Different population studies have reported that the rates of cardiovascular mortality in HCV-positive patients were either similar6, 7 or higher,8 compared with the general population. Furthermore, two recent articles have reported that HCV seropositivity was an independent predictor for increased coronary atherosclerosis,9, 10 although other studies failed to identify HCV infection as a risk factor for acute myocardial infarction.11, 12 Finally, intima-media thickness (IMT) and the prevalence of carotid artery plaques were found to be either increased13-17 or normal11 in HCV patients, and genomic and antigenomic HCV RNA strands were identified within carotid plaque tissues.13, 18
This evidence is in keeping with a possible higher-than-normal cardiovascular risk in HCV patients. However, these studies did not directly evaluate the relative role of viral and metabolic factors in determining cardiovascular risk. In addition, these studies were performed in patients with a clinical diagnosis of CHC, and no data are available on the potential associations between cardiovascular risk and the histological grading and staging of liver disease (i.e., necroinflammatory activity, fibrosis, and steatosis).
We evaluated the prevalence and severity of carotid atherosclerosis in a homogeneous cohort of biopsy-proven G1 CHC patients, compared with a control population, in order to assess the potential role of host and viral factors, including liver histology, in vascular abnormalities.
In all, 174 consecutive patients with G1 CHC were prospectively recruited at the Gastrointestinal & Liver Unit of the Palermo University Hospital. All patients fulfilled the inclusion and exclusion criteria detailed below. Patients were included if they had a histological diagnosis of CHC (any degree of fibrosis, including cirrhosis) on a liver biopsy performed within 6 months prior to enrollment. G1 CHC patients were characterized by the presence of anti-HCV and HCV RNA, with persistently abnormal alanine aminotransferase (ALT), and by alcohol consumption <20 g/day in the past year or more, evaluated by a specific questionnaire.
Exclusion criteria were: (1) advanced cirrhosis (Child-Pugh B and C); (2) hepatocellular carcinoma; (3) liver disease of different or mixed etiology (i.e., excessive alcohol consumption, hepatitis B, autoimmune liver disease, Wilson's disease, hemochromatosis, α1-antitrypsin deficiency); (4) HIV infection; (5) previous treatment with antiviral therapy, immunosuppressive drugs, and/or regular use of steatosis-inducing drugs (corticosteroids, valproic acid, tamoxifen, amiodarone); (6) use or previous use of drugs interfering with lipid metabolism (i.e., statins, fibrates); (7) previous diagnosis of carotid atherosclerosis; or (8) active intravenous illicit drug addiction.
Sixty-three consecutive G1 CHC patients observed in the same period of enrollment were ruled out due to absence of histological diagnosis (27/63), previous antiviral therapy (18/63), alcohol consumption greater than 20 g/day (7/63), previous diagnosis of carotid atherosclerosis (5/63), and statin use (6/63).
In all, 174 patients, matched for sex, age, and body mass index (BMI) with the CHC population were enrolled as controls. They were selected from a database of more than 1,000 asymptomatic patients referred by their general practitioners to the cardiology outpatient department of our hospital for a vascular screening of the carotid branch by ultrasonography. In particular, no subjects had a previous history of symptomatic cardiovascular disease (transient ischemic attack [TIA], stroke, angina, myocardial infarction, right or left hearth decompensation), had no previous instrumental diagnosis of atherosclerotic disease and/or of dilatative or hypertrophic heart disease, and were not taking drugs interfering with lipid metabolism. This population was considered as a group at low risk of atherosclerosis. All had normal ALT values (<37 IU/L), had no evidence of viral infection (anti-HCV, anti-HIV, and hepatitis B surface antigen [HBsAg] negativity), and reported an alcohol consumption <20 g/day during the previous year (evaluated by a specific questionnaire). Biochemical analyses were performed in the same central laboratory used for CHC patients, and history data were obtained using a standardized interview in both cases and controls.
The study was performed in accordance with the principles of the Declaration of Helsinki and its appendices and with local and national laws. Approval was obtained from the hospital's Institutional Review Board and Ethics Committee and written informed consent was obtained from all patients.
Clinical and Laboratory Assessment.
Clinical and anthropometric data were collected at the time of liver biopsy. BMI was calculated on the basis of weight, in kilograms, and height, in meters. Patients were classified as normal weight (BMI, 18.5-24.9 kg/m2), overweight (BMI, 25-29.9), or obese (BMI ≥30). The diagnosis of arterial hypertension was based on the following criteria: systolic blood pressure ≥135 mm Hg and/or diastolic blood pressure ≥85 mm Hg (measured three times within 30 minutes, in the sitting position, using a brachial sphygmomanometer), or use of blood-pressure-lowering agents. The diagnosis of type 2 diabetes was based on the revised criteria of the American Diabetes Association using a value of fasting blood glucose ≥126 mg/dL on at least two occasions.19 In patients with a previous diagnosis of type 2 diabetes, current therapy with insulin or oral hypoglycemic agents was documented.
A 12-hour overnight fasting blood sample was drawn at the time of biopsy to determine serum levels of ALT, total cholesterol, high-density lipoprotein (HDL) and LDL-cholesterol, triglycerides, plasma glucose concentration, insulin, and platelet count. Insulin resistance was determined with the homeostasis model assessment (HOMA).20
All patients were tested at the time of biopsy for HCV-RNA (homemade reverse-transcription polymerase chain reaction [RT-PCR]; limit of detection: 12 IU/mL). Genotyping was done with INNO-LiPA, HCV II (Bayer).
Slides were coded and read by one pathologist (D.C.), who was unaware of patient identity and history. A minimum length of 15 mm of biopsy specimen or the presence of at least 10 complete portal tracts was required.21 Biopsies were classified according to the Scheuer scoring system.22 The percentage of hepatocytes containing macrovescicular fat was determined for each 10× field. An average percentage of steatosis was then determined for the entire specimen. Steatosis was assessed as the percentage of hepatocytes containing fat droplets (minimum 5%) and evaluated as a continuous variable. Steatosis was classified as absent/mild at <30%, or moderate/severe at ≥30%.
Carotid Artery Evaluation.
Carotid atherosclerosis was evaluated by an expert physician (G.F.) of the cardiology department in a blinded fashion as to patient group using a high-resolution B-mode ultrasonography equipped with a multifrequency linear probe. The carotid arteries were investigated in longitudinal projections of both the left and right side at the level of the common carotid artery, bulb, and internal carotid in each patient. The carotid IMT was measured as the difference between the first (intima lumen) interface and the second (media adventitia) interface on the far wall of the common carotid artery in a section free of plaque beginning 10 mm below their bifurcations and including the bifurcations for 10 mm. For each subject, three measurements on both sides were performed, i.e., the anterior, lateral, and posterior projection of the near and far wall. Maximum (outside the plaque) rather than mean values of IMT were considered, and edge detection was performed manually. IMT measurements from the left and right side were averaged. A carotid plaque was defined as a focal thickening of >1.3 mm at the level of common and internal carotid arteries and bifurcations.
All ultrasound data for both cases and controls were recorded in the patient file and in an electronic database.
Because the ultrasonographic procedure for plaque evaluation and IMT measurement is highly subjective, we tested the intraobserver agreement in the last 30 cases included in the analysis after a median of 75 days.
Continuous variables were summarized as mean ± standard deviation (SD) and categorical variables as frequency and percentage. Student's t test and chi-square test were used when appropriate.
The planned sample size for this study was 360 patients with equal allocation to the case and control groups (180 per group). The sample size estimates were based on the expected proportion of cases and controls with carotid plaques. Sample size was calculated to detect a difference in carotid plaque prevalence of 15% between cases and controls. The sample size calculation was performed with an expected prevalence of plaques of 20% in the control group and 35% in the experimental group, using a two-sided test with a α error of 5% and a β error of 10%.
Multiple logistic regression models were used to assess the relationship of carotid plaques with other clinical, biochemical, and histological parameters in the entire cohort comprising both cases and controls and separately in G1 CHC patients. In these models the dependent variable was the presence of carotid plaque, coded as 1 versus 0 = absent. As candidate risk factors, we selected age, gender, BMI, baseline ALT, triglycerides, total and HDL cholesterol, blood glucose, diabetes, arterial hypertension, smoking, platelet count levels, and HCV infection (for the entire cohort). Insulin and HOMA, log HCV RNA, steatosis, necroinflammatory activity, and fibrosis scores were added in the CHC model.
Multiple linear regression analysis was done to identify independent predictors of IMT (continuous dependent variable) in CHC patients and in the entire cohort of both cases and controls. As candidate risk factors we selected the same independent variables included in the logistic models.
Variables associated with the dependent variable at univariate analysis (probability threshold, P ≤ 0.10) were included in the multivariate regression models. Regression analyses were done using Proc Logistic, Proc Reg, and subroutine in SAS (SAS Institute, Cary, NC).23
Patient Features and Histology.
The baseline features of the 174 patients are shown in Table 1. The mean age was 53 years (range 23-75 years). More than 50% of our patients were in the overweight to obesity range and a quarter of them were hypertensive. Diabetes was present in 7.5% of patients. Mean values for total cholesterol, HDL cholesterol, and triglycerides were within the normal range, whereas the mean HOMA values were elevated (3.25). One patient in three had fibrosis ≥3 by the Scheuer score, with a high prevalence of moderate/severe necroinflammation (grading 2-3). Half of the cases had histological evidence of steatosis, although of moderate/severe grade in only 38 cases (21.8%).
Table 1. Baseline Demographic, Laboratory, Metabolic Features of 174 Patients with Genotype 1 Chronic Hepatitis C and 174 Sex, Age, and BMI-Matched Controls
Genotype 1 Chronic Hepatitis C
(n = 174)
(n = 174)
IU, international units; HOMA, homeostasis model assessment; LDL, low density lipoprotein; HDL, high density lipoprotein; HCV-RNA, hepatitis C virus ribonucleic acid; N.A., not available. Data are given as mean ± standard deviation or as number of cases (%).
Mean age, years
53.2 ± 11.0
52.6 ± 9.7
Mean body mass index, kg/m2
26.2 ± 4.6
26.1 ± 3.6
Body mass index, kg/m2
Type 2 diabetes
Carotid intima-media thickness, mm
1.04 ± 0.21
0.90 ± 0.16
Alanine aminotransferase, IU/L
97.1 ± 87.8
26.9 ± 5.5
Platelet count X 103/mmc
204.4 ± 63.2
65.0 ± 72.3
177.7 ± 33.8
162.9 ± 35.2
HDL cholesterol, mg/dL
55.6 ± 17.8
44.9 ± 12.8
LDL cholesterol, mg/dL
100.9 ± 33.4
100.9 ± 29.7
96.4 ± 42.4
92.1 ± 55.4
Blood glucose, mg/dL
97.6 ± 35.5
93.2 ± 22.3
13.3 ± 7.6
3.25 ± 2.31
5.76 ± 0.71
Histology at biopsy
Steatosis - %:-continuous variable
13.6 ± 18.7
≥5% to <30%
Stage of fibrosis
Grade of inflammation
Control patients were comparable for gender (75 men), age (52.6 ± 9.7 years; P = 0.54), and BMI (26.1 ± 3.6 kg/m2; P = 0.77) to CHC patients. In addition, no differences were observed in LDL-cholesterol levels (100.9 ± 29.7 mg/dL; P = 0.89), presence of arterial hypertension (48 patients; P = 0.81), type 2 diabetes (16 patients; P = 0.56), and smokers (52 patients; P = 0.90). Nonetheless, the control patients had much lower ALT (26.9 ± 5.5 IU; P < 0.001) and HDL-cholesterol levels (44.9 ± 12.8 mg/dL; P < 0.001), and higher total cholesterol (162.9 ± 35.7 mg/dL; P < 0.001) compared with HCV-infected patients.
Factors Associated with Carotid Atherosclerosis.
Carotid plaques were found in 73 (41.9%) G1 CHC patients compared with 40 (22.9%) control patients (P < 0.001). Similarly, G1 CHC patients had a greater maximum IMT compared with control patients (1.04 ± 0.21 versus 0.91 ± 0.11; P < 0.001). None of the study participants had clinically relevant carotid stenosis (i.e., ≥60%).
In G1 CHC patients, older age (P = 0.01), low platelet count (P = 0.005), and arterial hypertension (P = 0.001) were associated with higher maximum IMT, although only low platelet count (P = 0.04) and arterial hypertension (P = 0.01) were independent factors at multiple linear regression analysis. Similarly, in the entire cohort including both cases and controls, older age (P < 0.001), arterial hypertension (P < 0.001), and HCV infection (P < 0.001) were independently linked to higher IMT by multivariate linear regression analysis. Similar results were obtained in all the models when the maximum IMT was replaced by mean IMT was used of individual patients as a linear dependent variable.
The univariate and multivariate comparisons of variables between G1 CHC patients with and without carotid plaques are reported in Table 2. Older age, moderate/severe necroinflammatory activity, and severe fibrosis were associated with the presence of carotid plaques (P < 0.10), even if only older age (odds ratio [OR] 1.047, 95% confidence interval [CI]: 1.014-1.082, P = 0.005) and severe fibrosis (OR 2.177, 95% CI: 1.043-4.542, P = 0.03) were the factors independently linked to the presence of carotid plaques at multivariate logistic regression analysis. Interestingly, no association was found between steatosis and the presence of carotid plaques. Receiver operator characteristic (ROC) curves analysis identified an age of >55 years (area under the curve [AUC], 0.655; standard error [SE], 0.041; 95% CI: 0.574-0.736; sensitivity, 64%; specificity, 62%) as the best cutoff for predicting the presence of carotid plaques. At this cutoff, 26/88 (29.5%) patients ≤55 years had carotid plaques compared with 47/86 (54.6%) >55 (P = 0.001).
Table 2. Univariate and Multivariate Analysis of Risk Factors Associated with the Presence of Carotid Plaques in 174 Patients with Genotype 1 Chronic Hepatitis C by Logistic Regression Analysis
No Carotid Plaques
Univariate Analysis P value
Multivariate Analysis OR (95% CI) P value
n = 101
n = 73
IU, international units; HOMA, homeostasis model assessment; HDL, high density lipoprotein; HCV-RNA, hepatitis C virus ribonucleic acid. Data are given as mean ± standard deviation or as number of cases.
50.7 ± 11.5
56.8 ± 9.3
1.047 (1.014, 1.082) 0.005
Body mass index, kg/m2
26.6 ± 4.9
26.1 ± 4.4
Type 2 diabetes
Alanine aminotransferase, IU/L
95.1 ± 95.0
99.8 ± 77.4
Platelet count X 103/mmc
206.4 ± 77.0
61.4 ± 58.6
70.1 ± 88.3
176.6 ± 35.4
179.3 ± 31.7
HDL cholesterol, mg/dL
56.7 ± 18.6
54.1 ± 16.8
LDL cholesterol, mg/dL
99.1 ± 33.9
103.3 ± 32.9
97.8 ± 48.7
94.4 ± 31.7
Blood glucose, mg/dL
95.4 ± 32.7
100.7 ± 39.1
13.5 ± 7.5
13.1 ± 7.8
3.15 ± 2.02
3.40 ± 2.67
5.71 ± 0.79
5.83 ± 0.57
Histology at biopsy
13.0 ± 18.5
14.5 ± 19.0
Grade of inflammation
0.756 (0.327, 1.748) 0.51
Stage of fibrosis
2.177 (1.043, 4.542) 0.03
When age was used in the model as a categorical variable at a threshold of 55 years, severe fibrosis (OR 2.234, 95% CI: 1.072-4.657 P = 0.03) and age of >55 years (OR 2.389, 95% CI: 1.246-4.579, P = 0.009) remained significantly associated with the presence of carotid plaques.
Of note, patients ≤55 years and with F0-F2 fibrosis had a prevalence of carotid plaque of 22.3% (15/67), significantly lower than the 52.3% (11/21) observed in patients with F3-F4 fibrosis (P = 0.008) (Fig. 1). In contrast, in patients >55 years the prevalence of carotid plaques was similar in those with or without severe hepatic fibrosis (25/43, 58.1% versus 22/43, 51.1%; P = 0.51) (Fig. 1).
In the entire cohort comprising both cases and controls, older age, high ALT, high total cholesterol, arterial hypertension, type 2 diabetes, and HCV infection were associated with the presence of carotid plaques (P < 0.10), but only older age (OR 1.070, 95% CI: 1.040-1.102, P < 0.001), high cholesterol levels (OR 1.010, 95% CI: 1.003-1.017, P = 0.005), type 2 diabetes (OR 3.494, 95% CI: 1.458-8.377, P = 0.005), and HCV infection (OR 2.105, 95% CI: 1.187-3.734, P = 0.01) were independent factors by multiple logistic regression analysis.
Finally, in the group of 30 cases with duplicate scans obtained by the same operator, the prevalence of carotid plaques did not differ and the mean percentage difference and its SD were 6.8% and 9.7%, respectively.
We found that patients with G1 CHC had a higher prevalence of carotid atherosclerosis compared with a control population, and identified older age and the presence of severe hepatic fibrosis as two factors independently associated with the presence of carotid plaques.
Conflicting literature data have reported either normal or increased IMT, and normal11 or increased13-17 prevalence of carotid artery plaques in patients with a clinical diagnosis of HCV infection compared with control populations. In keeping with a potential association between HCV infection and atherosclerotic disease, two recent articles9, 10 have highlighted that HCV seropositivity was an independent predictor of increased coronary atherosclerosis. In our study of patients with histologically diagnosed G1 CHC, we found that G1 CHC patients had higher IMT compared with a comparable control population of outpatients who received an ultrasonographic assessment of the carotid branch in a cardiology unit. In addition, we found that G1 CHC patients had a prevalence of carotid plaques around 40%, a value higher than that observed in the control population, and in line with the range of 38% to 64% reported in the literature for HCV-infected patients.14, 15 The significance of our results is further strengthened by the finding that HCV infection was identified as a factor significantly and independently associated with atherosclerotic disease, accounting for higher IMT and higher prevalence of carotid plaques.
This is the first study measuring carotid atherosclerosis carried out in histologically characterized G1 CHC patients. The novel finding is the independent association of the presence of carotid plaques with severe hepatic fibrosis, after adjustment for age. Similarly, we also found an independent association of IMT with low platelet count, an expression of more advanced fibrosis.
Although this study was merely observational and not designed to explore the reasons for the association of the prevalence of atherosclerosis with severe fibrosis in G1 CHC, some hypotheses can be proposed. No direct association was found between viral load and atherosclerosis, probably due to the fluctuating levels of viremia in HCV infection,24 although a direct role of HCV in the development of the atherosclerotic lesions might be suggested. Recent studies have demonstrated the presence of genomic and antigenomic HCV RNA strands within carotid plaque tissues in HCV-infected patients.13, 18 In addition, the proinflammatory and profibrogenic environment prompting fibrogenesis in the liver of HCV-infected patients could also be systemically activated, enhancing the development of atherosclerotic lesions, explaining the higher prevalence of carotid plaques in this subgroup of CHC patients.
We found that only 20% of younger patients (≤55 years) without severe fibrosis had carotid plaques, compared with 50% in the group with severe fibrosis. This finding is similar to the value observed in the subgroup of G1 CHC patients >55 years (again, about 50%), irrespective of the severity of fibrosis. Thus, severe fibrosis and the associated cascade of proinflammatory and profibrogenic pathways generated in the liver might promote carotid atherosclerosis at a much younger age, although this hypothesis needs validation in larger prospective studies also measuring serum levels and liver expression of proinflammatory and profibrogenic cytokines.
From a clinical point of view, our data indicate that HCV patients age 55 or more, as well as those with severe fibrosis, should undergo ultrasonography screening for carotid atherosclerotic disease. A recent study8 showed that HCV-positive blood donors have increased liver-related and cardiovascular mortality with respect to their noninfected counterparts, providing a rationale for proposing a vascular screening.
Finally, we could not identify any association between IMT, or the presence of carotid plaques, and traditional cardiovascular risk factors (e.g., high LDL, low HDL, diabetes, and hypertension), like observed when considering also the control population. This finding is intriguing. We can only hypothesize that in our patients, who had normal mean cholesterol values (partly due to a specific effect of HCV infection),25 most of the burden of traditional risk factors was cancelled out, and the presence of severe hepatic fibrosis thus becomes the leading predictive factor of atherosclerotic lesions.
The main limitation of this study lies in its case-control nature, where a selection bias in the recruitment of the control population could affect the interpretation of results. We enrolled as controls a population with similar risk factors (age, sex, and BMI), and a low a priori risk of cardiovascular damage. The prevalence of subclinical atherosclerosis in this group was similar to what was observed in other low risk populations.26-29 This strengthens the clinical significance of our results, showing that atherosclerotic lesions in low-risk HCV-infected patients are nonetheless more common and severe than those observed in a non-HCV-infected population at similar atherosclerotic risk.
A further methodological question is the potentially limited external validity of the results for different populations and settings. Our study included a cohort of European HCV patients, largely overweight, who were enrolled in a tertiary referral center for liver disease, limiting the broad application of the results. In addition, lack of data about the prevalence of liver steatosis in the control population could also affect the interpretation of our results.
In conclusion, the presence of advanced hepatic fibrosis identifies a subgroup of G1 CHC patients at higher risk of atherosclerotic lesions who should be carefully monitored in liver units, independently of their metabolic profile.
S. Petta designed the study, contributed to data acquisition, was responsible for writing the article, and participated in statistical analysis. C. Cammà, V. Di Marco, G. Parrinello, G. Licata, S. Novo, G. Marchesini, and A. Craxì (Director of the GI & Liver Unit) were responsible for the project and writing of the article. A. Mazzola, D. Cabibi, G. Fazio, A. Licata, and D. Torres participated in patient management and data collection. All authors have seen and approved the final version of the article.