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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References

Aim : To evaluate the effects of minimal to moderate alcohol consumption on the severity of histological lesions in patients with chronic hepatitis C.

Methods : Daily alcohol intake (none, 1–20, 21–30, 31–50 g/day) and histological activity and fibrosis were recorded in 260 patients with chronic hepatitis C.

Results : The proportion of patients with moderate (A2) or marked (A3) activity increased gradually from 53.8% in abstinent patients to 86.5% for an intake between 31 and 50 g/day (P = 0.003). In multivariate analysis, age > 40 years, alcohol intake between 31 and 50 g/day and moderate or severe steatosis were independently related to histological activity. The proportion of patients with moderate (F2) or marked (F3) fibrosis or cirrhosis (F4) gradually increased from 29.0% in abstinent patients to 67.6% for an intake between 31 and 50 g/day (P < 0.001). Multivariate analysis also showed that alcohol intake between 31 and 50 g/day, moderate or severe steatosis and histological activity were independently related to fibrosis. The deleterious effect of alcohol intake on histological lesions differed according to gender.

Conclusions : This study demonstrates that both activity and fibrosis gradually increase according to the amount of alcohol ingested, and that even moderate alcohol consumption, as low as 31–50 g/day in men and 21–50 g/day in women, may aggravate histological lesions in patients with chronic hepatitis C.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References

Hepatitis C virus (HCV) infection affects 1–3% of the population in industrialized countries, and a well-identified risk is progression to fibrosis and then cirrhosis.1–3 The rate of progression of chronic hepatitis C differs from one patient to another, and it is unclear why some patients develop severe fibrosis or cirrhosis, whereas others do not.4, 5 Several host, viral and environmental factors have been studied with regard to their impact on the progression of fibrosis, but their effects are controversial.6–8 A heavy alcohol intake of more than 50–60 g/day is probably a major environmental factor, and has been identified as an independent risk factor for progression of chronic hepatitis C to cirrhosis in numerous studies.6, 9–13 However, the threshold level of alcohol intake which negatively influences the natural course of HCV infection remains unknown, as does the impact of minimal to moderate (1–20, 21–30, 31–50 g/day) alcohol intake on the degree of histological lesions of the liver. Thus, the aim of this prospective study of a large cohort of consecutive patients with chronic hepatitis C was to evaluate the relationship between minimal to moderate alcohol intake and the degree of histological liver lesions.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References

Between November 1996 and April 1998, 260 consecutive patients seen in our department with histologically proven chronic hepatitis C were studied prospectively. At the time of the study, none of the patients had been treated for hepatitis C or consumed alcohol at more than 50 g/day. The baseline characteristics of the patients are shown in Table 1. There were 150 men and 110 women, with a mean age of 42.9 ± 12.2 years. The route of HCV transmission was blood transfusion in 78 cases (30.0%), intravenous drug use in 80 cases (30.8%) and unidentified in 102 cases (39.2%). The body mass index was determined in 181 patients. Both serum anti-HCV antibodies (second- and third-generation enzyme-linked immunoabsorbent assays, Ortho Diagnostic Systems, Raritan, NJ, USA) and serum HCV RNA (Amplicor HCV, Roche Molecular Systems, Pleasanton, CA, USA) were present in all patients. Serum HBsAg and anti-human immunodeficiency virus antibodies were absent. The HCV genotype was determined in 255 patients (INNO-LIPA HCV II, Innogenetics, Ghent, Belgium). The HCV RNA load was measured at the time of liver biopsy using a second-generation bDNA assay (Quantiplex HCV-RNA 2.0, Chiron Diagnostics, Emeryville, CA, USA) in 180 patients for whom frozen serum was available. All liver biopsies were fixed in formalin, embedded in paraffin and routinely processed for histological analysis. The grade of histological activity and the stage of histological fibrosis were assessed using the validated METAVIR scoring system, as follows: A1–A3 for the degree of necro-inflammatory activity (A1, mild activity; A3, marked activity) and F0–F4 for the degree of fibrosis (F0, absence of fibrosis; F4, cirrhosis).14, 15 Steatosis, graded as absent, mild (< 10% of hepatocytes), moderate (10–30% of hepatocytes) or marked (> 30% of hepatocytes), and histological lesions consistent with alcohol-induced liver disease (i.e. Mallory bodies and inflammatory infiltration predominantly composed of neutrophils) were recorded. In addition, a semi-quantitative histological grading in the range 0–3 (0, absent; 1, mild; 2, moderate; 3, marked) was used for the histological assessment of liver iron accumulation on Perls' staining. Alcohol intake was estimated on the basis of beverage-specific quantity frequency measures validated by the 1988 United States National Health Interview Survey.16 A questionnaire was given to each patient on the day of liver biopsy to determine the alcohol intake (number of drinks consumed per day), separately for beer, wine and spirits, on weekdays and at weekends, during the 6 months preceding evaluation. It was checked that, during this period, the alcohol intake was stable and was not influenced by the diagnosis of chronic hepatitis C. According to the level of alcohol intake, the patients were classified into four groups. Intake was considered to be minimal (1–20 g/day) in 96 patients (36.9%), mild (21–30 g/day) in 34 (13.1%) and moderate (31–50 g/day) in 37 (14.2%). Ninety-three patients (35.8%) denied alcohol intake and none of the patients consumed more than 50 g/day.

Table 1.  Baseline characteristics of the 260 patients with chronic hepatitis C
  • HCV, hepatitis C virus.

  • *

     During the 6 months before liver biopsy.

  •  Information lacking in five patients.

  •  Information available for 180 patients.

Mean age ± standard deviation (years) 42.9 ± 12.2
Gender
 Men150 (57.7%)
 Women110 (42.3%)
Route of HCV transmission
 Blood transfusion78 (30.0%)
 Intravenous drug use80 (30.8%)
 Not identified102 (39.2%)
Alcohol intake (g/day)* 10.4 ± 15.8
Histological activity (grade)
 A192 (35.4%)
 A2154 (59.2%)
 A3 14 (5.4%)
Fibrosis (stage)
 F0–F1162 (62.3%)
 F257 (21.9%)
 F331 (11.9%)
 F4 10 (3.9%)
Steatosis
 Absent85 (33.3%)
 Mild95 (37.3%)
 Moderate44 (17.3%)
 Marked31 (12.1%)
HCV genotype
 1, 2 or 4198 (77.6%)
 357 (22.4%)
Viral load (log 10 genome equivalent/mL)  6.2 ± 9.2

Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References

The distribution of qualitative variables was compared using the chi-squared test or Fisher's exact test, as appropriate. Mantel–Haenszel statistics were used to test the relationship of variables adjusted to a third variable. Quantitative variables were expressed as the mean ± standard deviation and were compared using the non-parametric Mann–Whitney test. A P value of less than 0.05 was considered to be significant. Multivariate analysis was used to identify independent factors related to histological activity and fibrosis by means of stepwise logistic regression analysis.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References

The relationship between alcohol intake and histological activity is shown in Figure 1. The proportion of patients with moderate or marked activity (A2–A3) increased gradually with the level of alcohol intake. Likewise, the proportion of patients with mild activity (A1) fell with increasing alcohol intake (Figure 1). Signs of alcohol-induced liver injury were carefully sought on liver biopsy specimens: only two patients had Mallory bodies (both consumed alcohol at 50 g/day) and none showed infiltration predominantly composed of neutrophils.

image

Figure 1. Relationship between histological activity (METAVIR scoring system) and the level of alcohol intake in patients with chronic hepatitis C. Grades A2–A3 were found in 50 of 93 patients (53.8%) who denied alcohol intake, 61 of 96 (63.5%) who had minimal intake, 25 of 34 (73.5%) who had mild intake and 32 of 37 (86.5%) who were moderate drinkers (P = 0.003).

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The proportion of patients with moderate or marked steatosis also increased gradually with the level of alcohol intake, from 20.2% (18 of 89 patients) in patients who denied alcohol intake to 54.1% (20 of 37 patients) in moderate drinkers (Figure 2). Moderate or marked steatosis was found more frequently in patients with a body mass index above 25 kg/m2 than in patients with a body mass index below 25 kg/m2 (40.3% vs. 20.2%, respectively; P < 0.001). The proportion of patients with moderate or marked histological activity (A2–A3) increased gradually with the degree of steatosis, from 36 of 85 patients (42.4%) without steatosis to 66 of 75 patients (88.0%) with moderate or marked steatosis (Figure 3). Moderate or marked steatosis was more frequent in patients infected by HCV genotype 3 than in patients infected by HCV genotypes 1, 2 or 4 (56.1% vs. 21.7%, respectively; P < 0.001).

image

Figure 2. Relationship between the level of alcohol intake and the presence of moderate or marked steatosis in patients with chronic hepatitis C. Moderate or marked steatosis was found in 20.2% (18 of 89 patients) of patients who denied alcohol intake, 26.0% (25 of 96 patients) of those with minimal intake, 36.4% (12 of 33 patients) of those with mild intake and 54.1% (20 of 37 patients) of those with moderate intake (P < 0.001).

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image

Figure 3. Relationship between histological activity (METAVIR scoring system) and steatosis in patients with chronic hepatitis C. Grades A2–A3 were observed in 36 of 85 patients (42.4%) without steatosis, 63 of 95 patients (66.3%) with mild steatosis and 66 of 75 patients (88.0%) with moderate or marked steatosis (P < 0.001).

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A significant relationship was found between the presence of liver iron accumulation and the degree of alcohol intake. Liver iron accumulation was significantly more frequent in patients with moderate and mild alcohol intake (53%) than in those with minimal intake (42%) or without intake (28%) (P < 0.05).

As confounding factors may explain the relationship between alcohol intake and histological activity, multivariate analysis was performed. The factors studied were the age at biopsy, gender, HCV transmission group, liver iron accumulation, steatosis, HCV genotype and alcohol intake. Age > 40 years [odds ratio (OR), 5.0; 95% confidence interval (CI), 1.8–13.0], alcohol intake between 31 and 50 g/day (OR, 6.6; 95% CI, 1.5–29.6) and moderate or marked steatosis (OR, 3.05; 95% CI, 1.0–9.6) were independently related to histological activity.

The relationship between the level of alcohol intake and the degree of histological fibrosis is shown in Figure 4. The proportion of patients with moderate or marked fibrosis or cirrhosis (F2–F3–F4) increased gradually with alcohol intake. The proportion of patients with mild or no fibrosis (F0–F1) decreased with increasing alcohol intake (Figure 4). A significant relationship was observed between the severity of fibrosis and the degree of steatosis. The proportion of patients with moderate or marked fibrosis or cirrhosis was 15.3% (13/85) in patients without steatosis and 62.7% (47/75) in patients with moderate or marked steatosis (Figure 5). The factors studied in the multivariate analysis were age at biopsy, gender, HCV transmission group, liver iron accumulation, steatosis, HCV genotype, histological activity and alcohol intake. Alcohol intake between 31 and 50 g/day (OR, 4.3; 95% CI, 1.2–16.0), moderate or marked steatosis (OR, 2.5; 95% CI, 1.0–6.5) and histological activity (OR, 4.9; 95% CI, 1.6–15.0) were independently related to histological fibrosis.

image

Figure 4. Relationship between histological fibrosis (METAVIR scoring system) and the level of alcohol intake in patients with chronic hepatitis C. Stages F2–F4 were observed in 27 of 93 patients (29.0%) who denied alcohol intake, 33 of 96 (34.4%) who had minimal intake, 13 of 34 (38.2%) who had mild intake and 25 of 37 (67.6%) who were moderate drinkers (P < 0.001).

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image

Figure 5. Relationship between fibrosis (METAVIR scoring system) and steatosis in patients with chronic hepatitis C. Stages F2–F4 were observed in 15.3% (13 of 85 patients) of patients without steatosis, 39.0% (37 of 95 patients) of those with mild steatosis and 62.7% (47 of 75 patients) of those with moderate or marked steatosis (P < 0.001).

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The effects of alcohol intake on histological activity and fibrosis are shown in Figures 6 and 7 according to gender. In women, alcohol intake of more than 20 g/day increased the degree of histological activity and fibrosis. The proportion of women with moderate or marked activity (A2–A3) was 63.3% for an alcohol intake of less than 20 g/day vs. 85.0% for an alcohol intake of more than 20 g/day (P = 0.06). Similarly, the proportion of women with moderate or marked fibrosis or cirrhosis (F2–F3–F4) was 31.1% for an alcohol intake of less than 20 g/day vs. 60.0% for an alcohol intake of more than 20 g/day (P = 0.02). The pattern was similar in men, but with a higher threshold dose than in women (30 g/day). The proportion of men with moderate or marked activity (A2–A3) was 57.0% for an alcohol intake of less than 30 g/day vs. 86.2% for an alcohol intake of more than 30 g/day (P = 0.003). Similarly, the proportion of men with moderate or marked fibrosis or cirrhosis (F2–F3–F4) was 32.2% for an alcohol intake of less than 30 g/day vs. 65.5% for an alcohol intake of more than 30 g/day (P = 0.004).

image

Figure 6. Relationship between histological activity (METAVIR scoring system) and the level of alcohol intake, according to gender, in patients with chronic hepatitis C.

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image

Figure 7. Relationship between histological fibrosis (METAVIR scoring system) and the level of alcohol intake, according to gender, in patients with chronic hepatitis C.

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Frozen serum samples were available for 180 patients; they were classified into three groups according to the level of alcohol intake (< 20, 21–30, 31–50 g/day) in order to study the relationship between alcohol intake and viral load. No significant difference was observed between the three groups. The viral loads (in log 10 genome equivalent/mL) were 6.2 ± 0.8, 6.3 ± 0.9 and 6.4 ± 0.9 for alcohol intakes of less than 20 g/day, 21–30 g/day and 31–50 g/day, respectively (N.S.). No relationship was found between viral load and the degree of histological activity or fibrosis (Table 2).

Table 2.  Viral load, histological activity and fibrosis (METAVIR scoring system) in 180 patients with chronic hepatitis C
 Viral load (log 10 genome equivalent/mL)
  1. P = N.S.

Histological activity
 A1 (n = 66)6.07 ± 0.8
 A2 (n = 105)6.29 ± 0.9
 A3 (n = 9)6.26 ± 0.7
Fibrosis
 F0–F1 (n = 119)6.10 ± 0.8
 F2 (n = 37)6.50 ± 0.8
 F3 (n = 16)6.18 ± 0.7
 F4 (n = 7)6.54 ± 0.7

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References

This prospective study of a large cohort of consecutive patients with chronic hepatitis C clearly demonstrates that moderate alcohol consumption, as low as 31–50 g/day in men and 21–50 g/day in women, may aggravate histological lesions.

With regard to histological activity, the proportion of patients with moderate or marked activity (A2–A3 in the METAVIR scoring system) gradually increased with the level of daily alcohol consumption (Figure 1). This increase in histological activity was related to an aggravation of HCV-induced liver lesions due to alcohol itself and not to the co-existence of HCV- and alcohol-induced liver lesions. Indeed, the histological lesions in drinkers, although more severe, were qualitatively similar to those seen in chronic hepatitis C. Multivariate analysis clearly demonstrated that even moderate alcohol intake (31–50 g/day) aggravates histological activity independently in patients with chronic hepatitis C. A recent study yielded conflicting results on the relationship between histological activity and alcohol intake. In that study, histological activity was related to past alcohol intake (before the diagnosis of HCV infection) and negatively related to present alcohol intake (1 week before hospitalization).11 The reason for the difference between that study and our results is not clear. It should be stressed, however, that past alcohol intake was evaluated at the diagnosis of HCV infection (a mean of 1 year before liver biopsy) and current alcohol intake was assessed during a short period before liver biopsy and was generally low (> 20 g/day in only 17% of patients).

In the present study, a significant link was also found between the degree of histological fibrosis and the level of alcohol intake. Indeed, the proportion of patients with moderate or marked fibrosis or cirrhosis (F2–F3–F4) gradually increased with daily alcohol intake (Figure 4). Multivariate analysis showed that even moderate alcohol intake (between 31 and 50 g/day) acted as an independent aggravating factor for fibrosis. In contrast, two large studies have demonstrated that only heavy alcohol intake (more than 50 g/day in women and 60 g/day in men) is an independent risk factor for the progression of fibrosis and cirrhosis.6, 17 Recently, past alcohol intake, evaluated a mean of 1 year before liver biopsy, was found to be significantly related to the severity of fibrosis,11 and total alcohol consumption, estimated over each subject's lifetime or throughout the duration of HCV infection, was identified as an independent factor associated with the presence of cirrhosis.12 One interesting observation in the present study was that the degree of fibrosis gradually increased with the level of alcohol consumption, and that heavy (> 50 g/day) and moderate (31–50 g/day) alcohol intake independently influenced the progression of fibrosis in patients with chronic hepatitis C. Another interesting observation was that, in women, an alcohol intake of more than 20 g/day increased both the histological activity and fibrosis, whereas in men the threshold value was 30 g/day. This result, obtained in patients with HCV infection, confirms previous findings in patients without HCV infection, namely that women have a higher risk than men of developing alcohol-induced liver disease,18, 19 and determines clearly for clinicians and patients the threshold level of alcohol intake which in practice negatively influences the course of chronic hepatitis C according to gender.

The mechanism of alcohol-induced aggravation of histological lesions of the liver due to HCV infection remains unclear. In theory, it could result from a direct effect of alcohol on HCV replication, alcohol-induced changes in the immune system or metabolic damage of liver cells caused by alcohol. Several studies have shown that HCV RNA serum levels are higher in patients who drink alcohol than in non-drinkers.9, 11, 20 However, this was not confirmed in our study, as viral load was not significantly influenced by alcohol consumption. This is consistent with the results of a study in which viral load was similar in patients who consumed more than 10 g/day alcohol and in those who consumed less than 10 g/day.9 Moreover, HCV RNA levels cannot explain the deleterious effects of alcohol on histological lesions of the liver, because it is now well established that the severity of liver lesions is not related to viral load. By contrast, alcohol-induced immunological changes might play a role. Experimental studies have suggested that chronic alcohol abuse induces transient T-suppressor cell dysfunction.21, 22 Likewise, in mice mmunized with HCV core DNA constructs, chronic ethanol feeding inhibits Th cell and cytotoxic T lymphocyte activity, and reduces cytokine expression. These effects were directly due to ethanol, as mice switched back to an isocaloric control diet recovered normal cellular immunity.23 Such changes in cellular immunity induced by alcohol have also been suggested in humans and, given the importance of immune function in the pathogenesis of chronic hepatitis C, they could contribute to the observed aggravation of histological lesions.

As expected, we found a relationship between the level of alcohol intake and the degree of steatosis, but an interesting finding was the relationship between histological lesions and steatosis. Indeed, as recently suggested,24, 25 the proportion of patients with moderate or marked activity and moderate or marked fibrosis gradually increased with the degree of steatosis (Figures 3 and 5). Furthermore, in our multivariate analysis, steatosis acted as an independent factor for both histological activity and fibrosis. The pathogenic link between steatosis and fibrosis remains unknown. However, steatosis could contribute to hepatocellular injury via lipid peroxidation.26–31 Steatosis is a frequent histological finding in patients with HCV infection and may be caused by various factors, including HCV itself,32, 33 obesity24, 25, 33 and alcoholism.33 The extent to which it contributes to the progression of the disease warrants further investigation.

In conclusion, this prospective study shows that alcohol intake significantly aggravates histological activity and fibrosis in patients with chronic hepatitis C. It demonstrates that both heavy (more than 50 g/day in women and 60 g/day in men) and moderate (21–50 g/day in women and 31–50 g/day in men) alcohol intake has a deleterious effect in this setting. The mechanism by which alcohol increases histological activity and fibrosis in chronic hepatitis C is unclear, although alcohol-induced steatosis seems to play a role. The effects of alcohol on viral replication are controversial, as is the role of alcohol-induced immunological changes in the onset and progression of liver lesions. In practice, even moderate alcohol intake should be avoided by patients with chronic hepatitis C.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Statistical analysis
  6. Results
  7. Discussion
  8. References