Alcohol is an important co-factor for both steatosis and fibrosis in Northern Italian patients with chronic hepatitis C

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1 Journal of Hepatology 41 (2004) Alcohol is an important co-factor for both steatosis and fibrosis in Northern Italian patients with chronic hepatitis C Paolo Fabris 1, *, Annarosa Floreani 2, Antonio Carlotto 3, Maria Teresa Giordani 1, Vincenzo Baldo 4, Clara Stecca 1, Lorella Marchioro 5, Andrea Tramarin 1, Tosca Bertin 6, Francesco Negro 7, Fausto de Lalla 1 1 Department of Infectious Diseases and Tropical Medicine, S. Bortolo Hospital, Viale Rodolfi 37, Vicenza, Italy 2 Department of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy 3 Department of Infectious Diseases, S. Camillo de Lellis Hospital, Schio, Vicenza, Italy 4 Department of Environmental Medicine and Public Health, Institute of Hygiene, University of Padova, Padova, Italy 5 Department of Pathology, S. Camillo de Lellis Hospital, Schio, Vicenza, Italy 6 Department of Gastroeneterology, S. Bortolo Hospital, Viale Rodolfi 37, Vicenza, Italy 7 Division of Gastroenterology and Hepatology and of Clinical Pathology, University Hospital Geneva, Switzerland Background/Aims: Steatosis in patients with chronic hepatitis C (CHC) may be the result of both viral and host factors. To evaluate: (1) the relationship between steatosis and either host or viral factors; (2) the correlation between steatosis and fibrosis in patients with CHC. Methods: A consecutive series of 349 patients were evaluated for steatosis. At liver biopsy, patients were tested for virological, and laboratory analysis and questioned for alcohol consumption. Results: Logistic regression analysis demonstrated that steatosis was independently associated with genotype 3a (odds ratio, OR 3.5), alcohol intake at the time of biopsy (OR 2.6) and age >35 years (OR 2.7). In multivariate analysis the presence of fibrosis was associated with past alcohol abuse (OR 3.7), and age older than 44 years (OR 2.2). Overall, a weak correlation was found between grade of steatosis and fibrosis score (rz0.861, PZ0.05), which disappeared excluding patients without past or current alcohol intake. A direct correlation emerged between grade of steatosis and both grading and staging only in patients with genotypes other than 3a. Conclusions: Genotype 3a is the main risk factor for steatosis in patients with CHC. The grade of steatosis correlated with both grading and staging only in patients with genotypes other than 3a and this relationship is strictly linked to alcohol consumption. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Chronic hepatitis C; Alcohol; Hepatic steatosis; Body mass index; Fibrosis; Genotypes 1. Introduction Nearly 170 million people worldwide have been infected with hepatitis C virus (HCV) [1,2]. In the majority of cases, chronic HCV infection is associated with chronic hepatitis, which can progress to liver cirrhosis and eventually to hepatocellular carcinoma. In long-term epidemiological Received 27 March 2004; received in revised form 25 May 2004; accepted 22 June 2004; available online 8 July 2004 * Corresponding author. Tel.: C ; fax: C address: pfabris@yahoo.com (P. Fabris). studies, up to 30% of patients with chronic hepatitis C (CHC) develop liver cirrhosis [3]. Many factors, e.g. age at infection, gender, hepatic iron content, and alcohol intake have been associated with disease progression in patients with chronic hepatitis C [4 7]. Steatosis, which is a common histological finding in patients with chronic hepatitis C infection, also seems to be a cofactor capable of contributing to hepatic fibrosis. Liver steatosis is characterized by intracellular fat accumulation and may be virus or host-related. In fact, there is increasing evidence that HCV may be an important cause of liver steatosis, supported by the higher prevalence of liver steatosis in /$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 P. Fabris et al. / Journal of Hepatology 41 (2004) patients with chronic hepatitis C than in the general population, and by the disappearance of fat droplets in patients responding to IFN treatment, particularly in those with genotype 3a [8 11]. Moreover, experimental studies have demonstrated that HCV core proteins can induce lipid droplets in transgenic mice [12,13]. On the other hand, host factors such as drugs, type 2 diabetes, obesity, iron accumulation and alcohol intake may lead to liver steatosis. In many patients with chronic hepatitis, steatosis appears to be multifactorial, due to two or more concomitant risk factors, though the relative weight of each factor is difficult to assess. Hourrigan et al. found an association between body mass index (BMI) and steatosis that correlated with the stage of fibrosis [14]. This association was recently confirmed in two large groups of patients in the United States [8,15]. Conversely, the relationship between BMI and steatosis proved weak in other studies, or associated mainly with viral genotype 1, stressing the influence of the different populations studied [16]. Understanding the relationships between host and virus factors is crucial to the management of patients with chronic hepatitis C. The aims of the present study were to evaluate the relationship between steatosis and host or viral factors in a large group of Italian patients with biopsy-proven chronic hepatitis C, and to assess the relationship between steatosis and fibrosis. 2. Patients and methods For this study we enrolled 349 patients consecutively biopsied for chronic hepatitis C at two different hospitals in north-east Italy (S. Bortolo Hospital, Vicenza and S. Camillo de Lellis Hospital, Schio). All patients were Italian-born and came from the north of Italy. They all had documented high serum aminotransferase of at least 6 months duration, they were positive for serum anti-hcv and HCV-RNA and negative for both HBsAg and HIV. Patients with autoimmune disorders or metabolic diseases (genetic hemochromatosis and Wilson s disease) as well as those previously treated with interferon or interferon plus ribavirin were ruled out. None of the patients had clinical or laboratory features of hepatic decompensation. Epidemiological and clinical data were obtained from all patients at the time of the liver biopsy. The body mass index (BMI, kg/m 2 ) was calculated for all patients and used to classify them as follows: normal weightzbmi!25; overweightzbmir25 but %30; obesezbmi>30. Details of alcohol consumption were obtained at the time of liver biopsy. For each patient, the alcohol intake (g/day) was calculated considering the last week before biopsy. Any alcohol consumption beyond 40 g/day for at least 5 consecutive years was also recorded. Liver biopsies were taken in patients who declared no alcohol intake or an alcohol intake!40 g/day, whereas it was deferred for at least 6 months in patients whose current alcohol consumption was >40 g daily. All patients who declared alcohol intake >40 g daily at the time of the first consultation were abstinent at the time of biopsy. Abstinence has been assessed by using validated scoring method [17]. For all patients, a fasting blood sample obtained on the day of liver biopsy was analyzed for the following parameters: glucose, cholesterol and triglycerides, HCV genotypes and HCV titer. Serum HCV-RNA viral titre was determined by using branched-dna signal amplification assay (HCV-bDNA 3.0, Bayer Diagnostics, Emeryville, CA). Hepatitis C virus genotypes were determined by nested PCR using genotype-specific primers of the core region and by DNA enzyme immunoassay DEIA, involving hybridization with subtype-specific probes Histological evaluation Histological evaluation was done by the same experienced pathologist with no information about the patient s clinical and virological data. Specimens were evaluated in terms of grading (necroinflammatory activity) and staging (fibrosis score) using Ishak s classification [18]. Steatosis was graded according to the percentage of hepatocytes affected. In detail: grade 0 (absent), grade 1 (!10%), grade 2 (10 30%), grade 3 (30 60%), and grade 4 (>60%) Statistical analysis Data were analyzed using the c 2 -test, Student s t-test for unpaired data and linear regression analysis, as appropriate. A P value %0.05 was considered significant and odds ratio (OR) with a 95% confidence interval (CI) was calculated for each parameters. A stepwise multiple regression analysis was performed for each variable under study to determine which characteristics were independently associated with steatosis and fibrosis. Analyses were performed using the Statistical Package for the Social Sciences (SPSS 11.0; SPSS Inc, Chicago, IL, USA). 3. Results The main characteristics of the patients are given in Table 1. Liver steatosis was observed in 213 cases (61%). Both the prevalence and the severity of liver steatosis were significantly higher (PZ and PZ0.0002, respectively) in patients with genotype 3a (77%, grade 3 4 in 36% of cases) than in cases with other genotypes (56%, grade 3 4 in 19% of cases,). The relationship between HCV genotypes and grade of liver steatosis is shown in Table 2.In addition to genotype 3a, univariate analysis showed a significant association between steatosis and obesity, alcohol consumption (being any alcohol intake at the time of biopsy or past alcohol abuse) hypertrygliceridemia, hypercholesterolemia, and age. No statistical association was found, on the other hand, between steatosis and the following variables: gender, viral titer, normal weight and overweight conditions, hyperglicaemia, and past intravenous drug use. Logistic regression analysis demonstrated that steatosis was statistically and independently associated with genotype 3a (OR 3.5, CI ), age classes (age between years OR 2.7, CI ; age >44 years, OR 2.1, CI ) alcohol intake at the time of biopsy (OR 2.6, CI ). The results of univariate and logistic regression analysis are shown in Table Relationship between steatosis and liver inflammation The histological inflammation grading was assessed for each parameter, as well as for total Hepatitis Activity Index (HAI) according to grade of steatosis. Overall, we found no significant correlation between the grade of steatosis and histological inflammation. In fact, mean total HAI score was 4.9G2.5 in patients without steatosis, and 5.5G2.4 in those with steatosis grade 3 4 (PZ0.07). Similar findings were found considering each parameter of Ishak classification (data not shown). However, different figures emerged when we stratified patients according to the HCV genotype.

3 646 P. Fabris et al. / Journal of Hepatology 41 (2004) Table 1 Clinical, virological and histological characteristics of the patients studied Characteristics N (%) Number of subjects 349 Mean agegsd (range) 39.6G12.9 (17 70) Gender (M/F) 269/80 Mean BMIGSD (range) 23.6G3.1(16 36) Normal (!25) 227 (65%) Overweight (R25 but %30) 108 (31%) Obese (O30) 14 (4%) Alcohol Past O40 g/day for more than 5 years 78 (22.3%) Current alcohol at the time of biopsy 66 (18.9%) IVDUs 190 (54.1%) Cholesterol (O200 mg/dl) 50 (14.3%) Glucose (O120 mg/dl) 19 (5.4%) Triglycerides (O170 mg/dl) 71 (20.3%) Viral load (O1.5!10 6 UI/ml) 43 (12.3%) Genotype 3a 90 (26%) 2a/c 77 (22%) 1a/1b 167 (48%) 4 15 (4%) Chronic hepatitis 325 (93%) Chronic hepatitis with cirrhosis 24 (7%) A significant increase, parallel to the grade of steatosis, was observed for interface hepatitis, focal apoptosis, as well as for total HAI score, in patients with genotypes other than 3a, but not in patients with genotype 3a (Table 4) Relationship between steatosis and fibrosis In our series, 243 patients (69%) had some degree of liver fibrosis with the following distribution: scores of 1 2 in 140 cases (58%), scores of 3 4 in 79 (32%), and scores of 5 6 in 24 (10%). In univariate analysis, the presence of fibrosis was significantly associated with both alcohol intake at the time of biopsy and past alcohol abuse, hypercholesterolemia, hypertriglyceridemia, age, and grade of steatosis. In multivariate analysis, the presence of fibrosis was independently and significantly associated with past alcohol abuse (OR 3.7, CI ), and age older than 44 years (OR 2.2, CI ) (Table 5). Overall, a weak correlation emerged between fibrosis score and grade of steatosis (rz0.861 PZ 0.05), Fig. 1. This trend disappeared, however, when the sub-group of 211 patients with no past or present alcohol intake was considered, demonstrating that alcohol is an important risk factor for both steatosis and fibrosis. A similar figure was obtained when all the potential risk factors considered for liver steatosis were excluded (alcohol, BMI>25, glycemia >120 mg/dl, triglycerides >170 mg/dl, cholesterol >200 mg/dl), Fig. 1. We also analyzed the relationship between grade of steatosis and fibrosis according to HCV genotype, comparing the 259 patients with HCV genotypes 1, 2 and 4 versus the group of 90 patients with genotype 3a (Fig. 2). The patients with genotypes 1, 2 and 4 were significantly older (mean age 42.1 versus 34.1 years, PZ0.04) and more frequently had a history of alcohol abuse (66/259, 25% versus 12/90, 13%, PZ0.03). By contrast, patients with genotype 3a more frequently had a history of intravenous drug use than patients with other genotypes (70/90, 78% versus 120/259, 46.3%, P!0.0001). A direct and significant correlation (R 1.00, PZ0.012) between grade of steatosis and mean fibrosis score emerged in patients with genotypes 1, 2 and 4, but not in patients with genotype 3a. Moreover, grade 3 4 steatosis was associated with a significantly higher fibrosis score in patients with genotypes other than 3a by comparison with patients with genotype 3a (mean fibrosis score 2.6G1.2 versus 1.5G0.7, PZ0.002) Fig Discussion This study analyzed the prevalence of steatosis and its relationship with inflammation and fibrosis in a large series of Italian patients with chronic hepatitis C coming from the same geographical area. The overall prevalence found in this study was 61%, which is comparable with findings reported in the USA [14,16] and Australia [15,19]. It is also comparable with the results reported in a histological study conducted in northern Italy [20], but rather higher than reported by Adinolfi et al. in southern Italy [9]. As reported by other authors, in both univariate and logistic regression analysis, genotype 3a was the most important variable associated with liver steatosis, confirming that fat accumulation is mainly due to the virus in this sub-group of patients. The steatosis associated with Table 2 Relationship between HCV genotypes and grade of steatosis Genotypes No. of cases Grade of steatosis n (%) Patients with steatosis n (%) 1a/1b (44.3) 72 (43.1) 21 (12.6) 93 (55.6) 2a/2c (42.8) 38 (49.3) 6 (7.9) 44 (57.1) 3a (23.3) 44 (48.9) 25 (27.8) a 69 (76.6) b (53.3) 6 (40.0) 1 (6.7) 7 (46.6) Total (39.0) 160 (45.8) 53 (15.2) 213 (61.0) a PZ0.0002, severity of steatosis (grade 3 4) in patients with genotype 3a versus severity of steatosis (grade 3 4) in patients with other genotypes. b PZ0.0006, steatosis in patients with genotype 3a versus other genotypes.

4 P. Fabris et al. / Journal of Hepatology 41 (2004) Table 3 Univariate and multivariate analysis: steatosis Univariate Steatosis Yes (nz213) No (nz136) OR 95% CI P Logistic regression Adjusted OR Adjusted 95% CI Low Upper Low Upper n (%) n (%) Gender (M/F) Male 166 (77.9) 103 (75.7) Female 47 (22.1) 33 (24.3) Group age!35 66 (31.0) 74 (54.4) (39.4) 30 (22.1) ! O44 63 (29.6) 32 (23.5) BMI Normal (!25) 130 (61.0) 97 (71.3) Overweight (S25 71 (33.4) 37 (27.2) but %30) Obese (O30) 12 (5.6) 2 (1.5) Alcohol for more than 5 years %40 g/die 153 (71.8) 118 (86.8) O40 g/die 60 (28.2) 18 (13.2) Alcohol at biopsy No 163 (76.5) 125 (91.9) Yes 50 (23.5) 11 (8.1) ! IVDUs No 100 (46.9) 59 (43.4) Yes 113 (53.1) 77 (56.6) Cholesterol (O200 mg/dl) No 171 (80.3) 128 (94.1) Yes 42 (19.7) 8 (5.9) ! Glucose (O120 mg/dl) No 198 (93.0) 132 (97.1) Yes 15 (7.0) 4 (2.9) Triglycerides (O170 mg/dl) No 156 (73.2) 122 (89.7) Yes 57 (26.8) 14 (10.3) ! Genotype Other 144 (67.6) 115 (84.6) 3a 69 (32.4) 21 (15.4) ! !0.001 Viral load (O1.5!10 6 mui/ml) No 182 (85.4) 124 (91.2) Yes 31 (14.6) 12 (8.8) OR, odds ratio; and CI, confidence interval. P genotype 3a was also more severe than the steatosis associated with other genotypes, and this fact appears consistent with previously-published papers [21,8]. However, at least two types of steatosis exist in patients with chronic hepatitis C, i.e. viral steatosis (associated mainly with genotype 3a) and the so-called metabolic steatosis, which includes steatosis related to type 2 diabetes, obesity, hyperinsulinism, drugs, and alcohol. Steatosis is a potentially reversible condition. Viral steatosis may disappear with antiviral treatment [22,23]. In metabolic steatosis, correcting the metabolic disorder may result in improved liver function tests and histology [24], while its persistence may accelerate the progression of fibrosis, and may reduce the likelihood of sustained virological response, particularly in patients infected with genotype 1 [22]. In contrast with other studies, and those performed in the USA and Australia in particular, we found no strong correlation between BMI and steatosis in patients with chronic hepatitis C [14,10,15]. These differences may have to do with the different populations studied. The mean age of our patients was 39 years and they had an average BMI of More than 50% of our patients had a history of intravenous drug use, while obesity and hyperglycemia only affected 4 and 6% of the sample, respectively much lower than in

5 648 P. Fabris et al. / Journal of Hepatology 41 (2004) Table 4 Relationship between grade of steatosis and liver inflammation according to Ishak s classification (data expressed as meangsd) in patients with genotype 3a and in patients with other genotypes Genotype 3a Other genotypes Grade 0 Grade 1 2 Grade 3 4 Grade 0 Grade 1 2 Grade 3 4 No of subjects Interface hepatitis 1.86G G G G0.95 a 1.72G G1.05 Confluent necrosis 0.86G G1, G G G G1.20 Focal apoptosis 1.38G G G G0.85 b 1.60G G0.77 Portal inflammation 1.95G G G G G G0.92 HAI total score 5.75G G G G2.29 c 5.62G G3.14 a Grade 0 versus grade 3 4: PZ b Grade 0 versus grade 3 4: PZ c Grade 0 versus grade 3 4: PZ other studies [10]. By contrast, we found a strong association between alcohol intake at the time of biopsy and liver steatosis both in univariate and in logistic regression analysis. This is consistent with the outcome of a study performed in Switzerland [25], but contrasts with many other studies performed in the USA and Australia [10,22,14]. A study recently reported by a French group ruled out heavy drinkers [21]. The study by Adinolfi et al. likewise excluded patients with an alcohol intake greater than 30 g/day [9]. Differences in the relative weight of alcohol in inducing steatosis in patients with chronic hepatitis C may therefore be related to methodological differences in measuring daily alcohol intake and establishing inclusion criteria, or to genuine differences in the daily alcohol consumption in different countries. Again, it may be difficult to establish how much alcohol a patient drinks, since many individuals tend to deny or underestimate their intake [26]. However, it is worth emphasizing that the present study was carried out in north-east Italy, where alcohol is an important social problem in terms of both morbidity and mortality. In Italy, alcohol consumption is higher in the north than in the south and mainly represented by wine drunk routinely with meals [27]. In a recent study by Bellentani et al. conducted in northern Italy, which excluded HBV and HCV patients, the prevalence of hepatic steatosis, evaluated by ultrasound scan, among heavy drinkers (30 g of alcohol a day) was 46.4% [28]. Based on the inclusion criteria for the present study, liver biopsies were taken in patients who reported no alcohol intake, or!40 g a day. Biopsy was deferred for at least 6 months in patients with an alcohol intake >40 g/day. Although the exact amount of daily alcohol intake capable of exerting a negative effect on the liver has not yet been defined, there is evidence that 20 g a day may be responsible for hepatic steatosis [29]. In patients with chronic hepatitis C and a moderate alcohol consumption, the histological pattern of alcoholic damage is usually unidentifiable [30]. We believe that this aspect has been underestimated in many studies or confounded by other variables [31]. The relationship between the grade of steatosis and inflammation remains controversial. Some authors have reported an association between these variables [14,9,21], whereas others were unable to reveal it [32,33]. Overall, we did not find any statistical association between the grade of steatosis and HAI score. However, when we stratified the population under study according to genotype, a significant increase, parallel to the grade of steatosis, was observed for interface Fig. 1. Overall correlation between grade of steatosis and fibrosis (A) in a subgroup of 271 patients with no history of present or past alcohol consumption (B) and in a subgroup of 144 patients with no risk factors for hepatic steatosis (C). [This figure appears in colour on the web.] Fig. 2. Relationship between grade of steatosis and mean fibrosis score according to HCV genotype. [This figure appears in colour on the web.]

6 P. Fabris et al. / Journal of Hepatology 41 (2004) Table 5 Univariate and multivariate analysis: fibrosis Univariate Fibrosis Yes (nz243) No (nz106) OR 95% CI P Logistic regression Adjusted OR Adjusted 95% CI Low Upper Low Upper n (%) n (%) Gender (M/F) Male 182 (74.9) 87 (82.1) Female 61 (25.1) 19 (17.9) Group age!35 82 (33.7) 58 (54.7) (35.4) 28 (26.4) O44 75 (30.9) 20 (18.9) BMI Normal (!25) 161 (66.3) 66 (62.3) Overweight (S25 74 (30.5) 34 (32.1) but %30) Obese (O30) 8 (3.3) 6 (5.7) Alcohol for more than 5 years %40 g/die 174 (71.6) 97 (91.5) O40 g/die 69 (28.4) 9 (8.5) ! Current alcohol at biopsy No 194 (79.8) 94 (88.7) Yes 49 (20.2) 12 (11.3) IVDUs No 116 (47.7) 43 (40.6) Yes 127 (52.3) 63 (59.4) Cholesterol (O200 mg/dl) No 199 (81.9) 100 (94.3) Yes 44 (18.1) 6 (5.7) Glucose (O120 mg/dl) No 229 (94.2) 101 (95.3) Yes 14 (5.8) 5 (4.7) Triglycerides (O170 mg/dl) No 182 (74.9) 96 (90.6) Yes 61 (25.1) 10 (9.4) Genotype Other 178 (73.3) 81 (76.4) 3a 65 (26.7) 25 (23.6) Viral load (O1.5!10 6 mui/ml) No 214 (88.1) 92 (86.8) Yes 29 (11.9) 14 (13.2) Steatosis Absent 80 (32.9) 56 (52.8) Grade (75.6) 39 (36.8) Grade (79.2) 11 (10.4) OR, odds ratio; and CI, confidence interval. P hepatitis, focal apoptosis, as well as for total HAI score, in patients with genotypes other than 3a, but not in patients with genotype 3a. The association between grade of steatosis and liver inflammation in patients with genotype different from 3a could be explained assuming that in this group of patients host factors play a prevalent role in inducing both steatosis and inflammation. On the contrary, in our experience, genotype 3a-associated steatosis, which has a prevalent viral etiology, seems to represent an innocent bystander. Since this finding is in contrast with previously published studies, this point needs to be confirmed in the future. The second aim of this study was to ascertain the relationship between steatosis and fibrosis. In 30% of liver biopsy examined fibrosis was absent. This could be explain again by the relative high number of patients with history of intravenous drug use and with a presumably short duration of infection.

7 650 P. Fabris et al. / Journal of Hepatology 41 (2004) Considering all patients, a significant association between steatosis and fibrosis was found only in the univariate analysis. Logistic regression analysis showed that fibrosis was significantly and independently associated with history alcohol abuse, age and obesity. However, by plotting pooled data in a linear correlation analysis a direct correlation between the grade of steatosis and fibrosis has emerged. Interestingly, using the same analysis, there was no correlation between stage of steatosis and fibrosis score when we considered the group of patients with no past or present alcohol intake, which further reinforces the convinction that alcohol is an important cofactor for both steatosis and fibrosis in our patients with chronic hepatitis C. This detrimental effect of alcohol on the natural history of chronic hepatitis C is well known [34], and the synergistic interaction between steatosis and even a low alcohol consumption could further contribute to the progression of liver fibrosis [35]. Furthermore, the correlation between the grade of steatosis and fibrosis score further decreased (particularly for grade 1 2 of steatosis) by plotting patients without any risk factor, confirming that fibrosis in patients with chronic hepatitis C is multifactorial. Indeed, the relationship between steatosis and fibrosis in our study seems to be closely linked to the HCV genotypes. A direct and significant correlation between grade of steatosis and fibrosis score was found in patients with genotypes 1, 2 and 4, but not in genotype 3a, despite the more severe steatosis in the latter. This finding is in agreement with Patton et al. [22] who, despite the higher prevalence and increased severity of steatosis, failed to find an association with the disease severity, as indicated by METAVIR fibrosis stage, in genotype 3 infected patients. This observation remains largely unexplainable. It could depend on a type beta error, on the effect of different enrolment criteria, or be the effect of other unidentified factors, such as dietary and genetic background. Metaanalysis on individual data pooled from all studies of sufficient quality may help to address this issue. Indeed, since these results come out from cross-sectional, not prospective studies, we cannot exclude the possibility that steatosis associated with genotype 3 represent a risk of fibrosis. Nevertheless, we must underline that in our study, patients with genotype 3 were younger than cases with the other genotypes. Most of them had a history of intravenous drug use, presumably with a shorter history of disease, normal body weight, and low alcohol consumption. Therefore, in this subgroup of patients, virus itself appears to be the main risk factor for steatosis. Whereas, in patients with other genotypes many host factors seem to prevail in inducing steatosis and influencing negatively the natural history of chronic hepatitis C, e.g. obesity, hyperlipemia and, most importantly, alcohol consumption. We believe that alcohol consumption is the most important factor affecting the natural history of chronic hepatitis C in our geographical area. It contributes heavily to inducing steatosis, liver inflammation and fibrosis. Our study suggests the need for adequate counseling in our region, focusing on reducing alcohol consumption, which would be extremely important in slowing the evolution of chronic hepatitis C. References [1] Alter MJ, Kruszon-Moram D, Naina OV, McQuillan GM, Gao F, Moyer LA, et al. 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