Fibrosis
2012
In a biopsy report, what does Stage mean, and what does Grade mean ?
“Stage” is the amount of fibrosis (scaring) detectable by biopsy…from stage one (mild) to four (cirrhosis). “Grade” is the amount of inflammation, which is caused by the activity of the virus. Generally speaking, inflammation is the precursor to fibrosis
When infected by the hepatitis C virus, liver cells (hepatocytes) trigger a series of events that initiates both an inflammatory and an immune response.
Two histological stages of chronic liver disease are fibrosis and cirrhosis.
Fibrosis is seen as scar formation in the patient's liver. The scars occur as the liver tries to repair damaged tissue. Cirrhosis is a condition where the liver becomes permanently scarred. Cirrhosis interferes with normal liver function, and many times, the patient is symptomatic.
Fibrosis
Fibrosis is an accumulation of fibrous tissue resulting from an imbalance between several types of liver cells. As liver cell structures change, the function of the liver is altered. Fibrosis is common to several chronic liver diseases, as it is a sign of hepatic injury. A diagnosis of fibrosis is usually determined with a liver biopsy or noninvasive tests .
There is a spectrum of liver injury seen. Necrosis indicates that liver cells have been damaged and died. If there is substantial damage one may see scar tissue forming to replace the dead liver cells. The term for this finding is fibrosis. If there is really a lot of liver damage, these scars may alter the structure of the liver forming nodules. This severe form of fibrosis is called
cirrhosis. In it's early stages, fibrosis is reversible, but cirrhosis, once developed, may be permanent.
Cirrhosis
When the liver becomes permanently injured and scarred, the condition is called cirrhosis. This chronic (long-term) disease results from slow deterioration of the liver. Damage to the structure of the liver causes the flow of blood to be blocked and slows liver function so that the liver cannot regulate the content of the blood and process nutrients, hormones, drugs, and
toxins (harmful substances) as well as it normally would.
In this article, we will provide an overview of some of the mechanisms of fibrosis as well as factors that contribute to the progression of fibrosis to cirrhosis.
Additionally, provided below is a 2006 study, which was published in the Journal of Viral Hepatitis, discussing how long it can take to progress from one stage of fibrosis to the next - once fibrosis is present.
The Key Players: Stellate and Kupffer Cells
Hepatic Stellate Cells
Under normal conditions, stellate ("star-shaped") cells are reservoirs of fat and vitamin A in the liver. They also contain filaments that can contract and regulate blood flow through the liver.
When activated by liver damage and the chemical by-products of inflammation, stellate cells transform and become capable of creating strands of collagen, a substance fundamental to the formation of scar tissue. These collagen strands are deposited in areas of inflammation in an effort to contain the spread of viral infection.
Kupffer Cells
Kupffer cells are specialized leukocytes (white blood cells) in the liver. They can move rapidly around the liver, and are responsible for the removal of particulate matter from the circulating blood, such as old or damaged red blood cells, bacteria, viruses, parasites and tumor cells.
When leukocytes from outside the liver are drawn to the area of infection by chemical signals called cytokines released by infected liver cells, they cooperate with Kupffer cells to produce chemical signals that cause stellate cells to begin producing collagen fibers.
In addition, Kupffer cells produce oxygen free-radicals. As we shall see, oxygen free-radicals play a major role in the progression and development of fibrosis.
The Pathophysiology of Fibrosis
The deposition of collagen in an area of injury is not an abnormal event. By attempting to enclose an injured or infected area with scar tissue, the body attempts to limit the spread of infection to other cells.
Normally, as an infection or injury resolves, the collagen matrix enclosing the injury is dissolved and activated stellate cells die off, allowing the tissue to return to normal.
Unfortunately, in a chronic illness such as hepatitis C infection, ongoing infection and inflammation causes the collagen matrix to grow more rapidly than it can be dissolved. The result is a surplus of scar tissue, which can eventually progress to cirrhosis.
The deposition of collagen has direct effects on liver function, specifically:
The Assessment of Fibrosis
Normally, a liver biopsy is performed to accurately assess the progression of fibrosis in liver disease. The following terms are often used to describe the changes in liver tissue associated with HCV infection:
Contributing Factors
Although the exact sequence of physical and chemical events leading to the development of fibrosis and cirrhosis is not precisely defined, researchers have identified factors which play important roles in the progression of disease.
Antioxidants
Normally, the liver is well equipped with a range of antioxidants. These are chemicals that can protect the liver from the damaging effect of oxygen free-radicals, which are byproducts of many cellular and metabolic processes.
However, in chronic liver disease, there appears to be a significant depletion of antioxidants. This is important because a surplus of oxygen free-radicals can create a condition known as oxidative stress, which has been associated with the progression of fibrosis.
Hepatic Iron Stores
Iron can accumulate in the liver as a result of genetic disease, such as hemochromatosis, or as the result of repeated blood transfusions. Iron overload is associated with liver injury, including fibrosis, cirrhosis and liver cancer.
A surplus of hepatic iron has been identified as a "pro-fibrogenic co-factor" in the presence of alcohol abuse, viral hepatitis, or hepatotoxic drugs.
Age
Age has been associated with increased vulnerability to the detrimental effects of oxidative stress, and this appears to be related to a decreased availability of antioxidant resources. Age is considered a significant determinant in the rate of progression from inflammation to fibrosis and cirrhosis.
Alcohol
Alcohol use has been shown to correlate highly with progressive fibrosis in the context of HCV infection, suggesting that abstinence from alcohol can reduce the rate of fibrotic progression to cirrhosis.
Obesity and NASH
Nonalcoholic steatohepatitis (NASH) is a serious liver disease that is characterized by fatty deposits in the liver and inflamed liver tissues. As we have seen, liver inflammation leads to scarring (fibrosis) and cirrhosis.
Steatosis (fatty liver), a precursor to NASH, is highly correlated with obesity. Steatosis has been found in 70% of people who exceed their ideal body weight by 10%, and in 100% of people who are morbidly obese.
Steatosis is usually a harmless and non-progressive condition, but in some people, steatosis develops into NASH. Studies have found that the incidence of NASH in obese people ranges from about 10% to 70% varying with age and degree of obesity.
Is There Treatment For Liver Fibrosis?
Obviously, the best way to prevent the progression of fibrosis / cirrhosis in the context of HCV infection is to eradicate the hepatitis C virus.
Unfortunately, peginterferon / ribavirin combinations fail to eliminate the virus in a percentage of patients, so anti-fibrotic therapies are under investigation.
Newer FDA approved therapies such as Telaprevir and Boceprevir used along with pegylated interferon/Ribavirin have a much higher success rate for clearing the virus in genotype 1 patients. *See the below article for new drugs currently in development for genotypes 1-4.
2012 Article;
Hepatitis C Drug Development Goes from Pony Ride to Rocket Launch
The in depth report includes the following topics; HCV drug resistance, DAA drug-drug interactions, nucleosides and nucleotide polymerase inhibitors, HCV protease inhibitors, the next generation of drugs, HCV quad therapy, genotypes 1-4, interferon free therapy and much more....
Related June 6, 2012 - Full Text - Reversibility of liver fibrosis
Rate of fibrosis progression
A study in the Journal of Viral Hepatitis, 2006, researchers found that the rate of fibrosis progression
was higher then once thought. Often treatment was delayed if the patient had no fibrosis, but if you read the summary below it may make you think twice about starting treatment before liver damage/fibrosis developes
Treating HCV in carriers with no/minimal liver fibrosis has been questioned mainly because this has been assumed to be a benign, stable condition with minimal risk of progression to 'clinically significant' liver disease......All our patients had the final liver biopsy taken more than 5 years after the initial one and almost two-thirds of them showed fibrosis progression, development of advanced fibrosis/cirrhosis being observed in 27% including more than one-third of those with F1 (portal fibrosis) in the initial biopsy.
These results clearly indicate that chronic hepatitis C is a progressive disease in many patients who initially present with no/minimal fibrosis and that progression to advanced fibrosis does occur in a significant number of cases within 5-10 years.....Progression to F3 or cirrhosis was seen in 36% of those with F1 initially.....Fibrosis is facilitated by older age and alcohol and associated with inflammatory activity and ALT levels.....By multivariate analysis high alcohol intake (>40 g/day) and steatosis correlated independently with fibrosis progression (data not shown)......The ALT profile during follow-up was also found to have a significant association with fibrosis progression. Thirty-one patients had persistently normal ALT during the entire follow-up period and nine of them (29%) showed fibrosis progression (3 from F0 to F1, 5 from F1 to F2 and 1 from F1 to F3) compared with 55 of 75 (73.3%) of those with elevated ALT.. Antiviral therapy should be considered in mild chronic hepatitis C."
.Fig. 1 Rate and grade of fibrosis progression seen in 106 patients with initially mild chronic hepatitis C.
In a biopsy report, what does Stage mean, and what does Grade mean ?
“Stage” is the amount of fibrosis (scaring) detectable by biopsy…from stage one (mild) to four (cirrhosis). “Grade” is the amount of inflammation, which is caused by the activity of the virus. Generally speaking, inflammation is the precursor to fibrosis
When infected by the hepatitis C virus, liver cells (hepatocytes) trigger a series of events that initiates both an inflammatory and an immune response.
Two histological stages of chronic liver disease are fibrosis and cirrhosis.
Fibrosis is seen as scar formation in the patient's liver. The scars occur as the liver tries to repair damaged tissue. Cirrhosis is a condition where the liver becomes permanently scarred. Cirrhosis interferes with normal liver function, and many times, the patient is symptomatic.
Fibrosis
Fibrosis is an accumulation of fibrous tissue resulting from an imbalance between several types of liver cells. As liver cell structures change, the function of the liver is altered. Fibrosis is common to several chronic liver diseases, as it is a sign of hepatic injury. A diagnosis of fibrosis is usually determined with a liver biopsy or noninvasive tests .
There is a spectrum of liver injury seen. Necrosis indicates that liver cells have been damaged and died. If there is substantial damage one may see scar tissue forming to replace the dead liver cells. The term for this finding is fibrosis. If there is really a lot of liver damage, these scars may alter the structure of the liver forming nodules. This severe form of fibrosis is called
cirrhosis. In it's early stages, fibrosis is reversible, but cirrhosis, once developed, may be permanent.
Cirrhosis
When the liver becomes permanently injured and scarred, the condition is called cirrhosis. This chronic (long-term) disease results from slow deterioration of the liver. Damage to the structure of the liver causes the flow of blood to be blocked and slows liver function so that the liver cannot regulate the content of the blood and process nutrients, hormones, drugs, and
toxins (harmful substances) as well as it normally would.
In this article, we will provide an overview of some of the mechanisms of fibrosis as well as factors that contribute to the progression of fibrosis to cirrhosis.
Additionally, provided below is a 2006 study, which was published in the Journal of Viral Hepatitis, discussing how long it can take to progress from one stage of fibrosis to the next - once fibrosis is present.
The Key Players: Stellate and Kupffer Cells
Hepatic Stellate Cells
Under normal conditions, stellate ("star-shaped") cells are reservoirs of fat and vitamin A in the liver. They also contain filaments that can contract and regulate blood flow through the liver.
When activated by liver damage and the chemical by-products of inflammation, stellate cells transform and become capable of creating strands of collagen, a substance fundamental to the formation of scar tissue. These collagen strands are deposited in areas of inflammation in an effort to contain the spread of viral infection.
Kupffer Cells
Kupffer cells are specialized leukocytes (white blood cells) in the liver. They can move rapidly around the liver, and are responsible for the removal of particulate matter from the circulating blood, such as old or damaged red blood cells, bacteria, viruses, parasites and tumor cells.
When leukocytes from outside the liver are drawn to the area of infection by chemical signals called cytokines released by infected liver cells, they cooperate with Kupffer cells to produce chemical signals that cause stellate cells to begin producing collagen fibers.
In addition, Kupffer cells produce oxygen free-radicals. As we shall see, oxygen free-radicals play a major role in the progression and development of fibrosis.
The Pathophysiology of Fibrosis
The deposition of collagen in an area of injury is not an abnormal event. By attempting to enclose an injured or infected area with scar tissue, the body attempts to limit the spread of infection to other cells.
Normally, as an infection or injury resolves, the collagen matrix enclosing the injury is dissolved and activated stellate cells die off, allowing the tissue to return to normal.
Unfortunately, in a chronic illness such as hepatitis C infection, ongoing infection and inflammation causes the collagen matrix to grow more rapidly than it can be dissolved. The result is a surplus of scar tissue, which can eventually progress to cirrhosis.
The deposition of collagen has direct effects on liver function, specifically:
- The presence of collagen fibers around individual hepatocytes impairs the cells ability to receive nutrition and results in shrinkage of the cell (hepatocellular atrophy).
- The accumulation of collagen fibers in the hepatic sinusoids (microscopic blood channels in the liver) obstructs the passage of substances from the blood to the hepatocytes, decreasing the liver's ability to remove drugs, toxins and metabolic waste products.
- Fibrosis around the veins of the liver and restricts blood flow, increasing vascular resistance and contributing to the development of portal hypertension. Portal hypertension, in turn, contributes to the development of esophageal varices, edema and ascites.
- Impaired blood flow through the liver forces arterial blood to bypass the filtering cells of the liver, further decreasing the efficiency of the liver and contributing to the death of hepatic cells.
The Assessment of Fibrosis
Normally, a liver biopsy is performed to accurately assess the progression of fibrosis in liver disease. The following terms are often used to describe the changes in liver tissue associated with HCV infection:
- Portal Inflammation: the portal areas are tiny tracts of connective tissue within the liver that contain branches of the portal vein, the hepatic artery and bile ducts.
- Piecemeal Necrosis: this term describes necrosis (cellular death) and inflammation around the portal areas.
- Fibrosis: the deposition of collagen fibers in the cell structure of the liver, forming scar tissue. The early stages of fibrosis are confined to the portal tracts.
- Bridging Fibrosis: an intermediate stage of fibrosis are characterized by expansion of collagenous (scar) tissue to the portal tracts and bridging between portal areas.
- Cirrhosis: a term used to describe significant deformation of the liver structure due to scarring.
Contributing Factors
Although the exact sequence of physical and chemical events leading to the development of fibrosis and cirrhosis is not precisely defined, researchers have identified factors which play important roles in the progression of disease.
Antioxidants
Normally, the liver is well equipped with a range of antioxidants. These are chemicals that can protect the liver from the damaging effect of oxygen free-radicals, which are byproducts of many cellular and metabolic processes.
However, in chronic liver disease, there appears to be a significant depletion of antioxidants. This is important because a surplus of oxygen free-radicals can create a condition known as oxidative stress, which has been associated with the progression of fibrosis.
Hepatic Iron Stores
Iron can accumulate in the liver as a result of genetic disease, such as hemochromatosis, or as the result of repeated blood transfusions. Iron overload is associated with liver injury, including fibrosis, cirrhosis and liver cancer.
A surplus of hepatic iron has been identified as a "pro-fibrogenic co-factor" in the presence of alcohol abuse, viral hepatitis, or hepatotoxic drugs.
Age
Age has been associated with increased vulnerability to the detrimental effects of oxidative stress, and this appears to be related to a decreased availability of antioxidant resources. Age is considered a significant determinant in the rate of progression from inflammation to fibrosis and cirrhosis.
Alcohol
Alcohol use has been shown to correlate highly with progressive fibrosis in the context of HCV infection, suggesting that abstinence from alcohol can reduce the rate of fibrotic progression to cirrhosis.
Obesity and NASH
Nonalcoholic steatohepatitis (NASH) is a serious liver disease that is characterized by fatty deposits in the liver and inflamed liver tissues. As we have seen, liver inflammation leads to scarring (fibrosis) and cirrhosis.
Steatosis (fatty liver), a precursor to NASH, is highly correlated with obesity. Steatosis has been found in 70% of people who exceed their ideal body weight by 10%, and in 100% of people who are morbidly obese.
Steatosis is usually a harmless and non-progressive condition, but in some people, steatosis develops into NASH. Studies have found that the incidence of NASH in obese people ranges from about 10% to 70% varying with age and degree of obesity.
Is There Treatment For Liver Fibrosis?
Obviously, the best way to prevent the progression of fibrosis / cirrhosis in the context of HCV infection is to eradicate the hepatitis C virus.
Unfortunately, peginterferon / ribavirin combinations fail to eliminate the virus in a percentage of patients, so anti-fibrotic therapies are under investigation.
Newer FDA approved therapies such as Telaprevir and Boceprevir used along with pegylated interferon/Ribavirin have a much higher success rate for clearing the virus in genotype 1 patients. *See the below article for new drugs currently in development for genotypes 1-4.
2012 Article;
Hepatitis C Drug Development Goes from Pony Ride to Rocket Launch
The in depth report includes the following topics; HCV drug resistance, DAA drug-drug interactions, nucleosides and nucleotide polymerase inhibitors, HCV protease inhibitors, the next generation of drugs, HCV quad therapy, genotypes 1-4, interferon free therapy and much more....
Related June 6, 2012 - Full Text - Reversibility of liver fibrosis
Rate of fibrosis progression
A study in the Journal of Viral Hepatitis, 2006, researchers found that the rate of fibrosis progression
was higher then once thought. Often treatment was delayed if the patient had no fibrosis, but if you read the summary below it may make you think twice about starting treatment before liver damage/fibrosis developes
Treating HCV in carriers with no/minimal liver fibrosis has been questioned mainly because this has been assumed to be a benign, stable condition with minimal risk of progression to 'clinically significant' liver disease......All our patients had the final liver biopsy taken more than 5 years after the initial one and almost two-thirds of them showed fibrosis progression, development of advanced fibrosis/cirrhosis being observed in 27% including more than one-third of those with F1 (portal fibrosis) in the initial biopsy.
These results clearly indicate that chronic hepatitis C is a progressive disease in many patients who initially present with no/minimal fibrosis and that progression to advanced fibrosis does occur in a significant number of cases within 5-10 years.....Progression to F3 or cirrhosis was seen in 36% of those with F1 initially.....Fibrosis is facilitated by older age and alcohol and associated with inflammatory activity and ALT levels.....By multivariate analysis high alcohol intake (>40 g/day) and steatosis correlated independently with fibrosis progression (data not shown)......The ALT profile during follow-up was also found to have a significant association with fibrosis progression. Thirty-one patients had persistently normal ALT during the entire follow-up period and nine of them (29%) showed fibrosis progression (3 from F0 to F1, 5 from F1 to F2 and 1 from F1 to F3) compared with 55 of 75 (73.3%) of those with elevated ALT.. Antiviral therapy should be considered in mild chronic hepatitis C."
.Fig. 1 Rate and grade of fibrosis progression seen in 106 patients with initially mild chronic hepatitis C.
Fibrosis progression in initially mild chronic hepatitis C: 36% with F1 Progress to F3 or cirrhosis
Journal of Viral Hepatitis, 2006, 13, 297-302
ABSTRACT
Summary.
The natural history of chronic hepatitis C presenting with no/minimal liver fibrosis is uncertain with controversies on risk of progression and need for antiviral treatment. We studied rates and determinants of fibrosis progression in initially mild chronic hepatitis C.
One hundred and six patients (mean age 41.65 ± 12.83 years) with chronic hepatitis C virus infection and no/minimal fibrosis in the initial liver biopsy (F0/F1 by METAVIR score) were followed prospectively while untreated with repeated biopsy after 5 or more years (mean interval 7.8 ± 1.51 years).
Patients showing fibrosis progression were compared with nonprogressors for baseline and follow-up parameters. Sixty-four patients (60.4%) showed fibrosis progression including 13 of 27 (49%) with F0 and 51 of 79 (65%) with F1.
Progression to F3 or cirrhosis was seen in 36% of those with F1 initially.
Fibrosis progression (ΔF/year) was associated with age (P < 0.0001), baseline and follow-up alanine aminotransferase (ALT) (P = 0.005), histological activity (P = 0.004) and steatosis (P = 0.002)
in the initial biopsy and use of alcohol (P = 0.008).
Thus liver fibrosis progression occurs in two-thirds of patients with initially mild chronic hepatitis C within 5-10 years and advanced fibrosis/cirrhosis develops in one-third of those with F1 initially. Fibrosis is facilitated by older age and alcohol and associated with inflammatory activity and ALT levels. Antiviral therapy should be considered in mild chronic hepatitis C.
Introduction
Chronic infection with the hepatitis C virus (HCV) affects around 170-200 million persons worldwide and is the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma in most western countries [1,2]. In recent years, end-stage liver disease caused by HCV has become the major reason for liver transplantation in the US and in Europe. Many studies have been conducted during the past decade with the aim of defining the natural history and outcomes of chronic HCV infection and the major determinants of liver disease progression [3].
These studies have been difficult to perform due to: (a) the often silent course of chronic hepatitis C that remains asymptomatic for decades even in cases who eventually progress to cirrhosis; (b) the slow rate of progression in many cases, taking several decades for development of clinically meaningful complications; (c) the great heterogeneity of the disease as influenced by viral and host variables; (d) the availability of antiviral therapies of increasing efficacy that has determined a progressive reduction in the proportion of untreated patients among those diagnosed to have chronic hepatitis C.
The earliest studies conducted in patients with a well recognized source of infection (post-transfusional or otherwise parenterally contaminated cases) seemed to indicate that only a minority of HCV infected patients develop cirrhosis over a 15-25-year period, the proportion being particularly low when patients infected at younger age were considered [4-6]. Overall these studies demonstrated rather low morbidity and mortality due to hepatitis C during the first two to three decades after infection and tended to describe the disease as benign and nonprogressive.
On the contrary, other studies conducted in patients presenting with long-lasting and advanced liver disease, reported high rates of liver related complications and deaths during rather short follow-up periods [7]. Currently, one of the most clinically relevant while still controversial issues relates to the identification of patients at significant risk of progression among those who are diagnosed with chronic HCV infection while still asymptomatic and with histologically mild liver disease.
Recent studies indicate that this condition is frequently observed in the general population [8] and is also one of the main presentation profiles of chronic HCV infection in clinical practice as these patients are referred for consultation after having been diagnosed at occasional testing for anti-HCV.
The current attitude is to take a liver biopsy in these cases and to assess prognosis on the basis of the fibrosis stage [2,9]. Patients with no (F0) or minimal fibrosis limited to the portal tracts (F1) are considered to have mild, nonprogressive liver disease while those with more advanced fibrosis (≥F2) are considered at significant risk of developing cirrhosis (F4).
Accordingly, antiviral therapy is usually strongly recommended in the latter cases while the indication to treat mild chronic hepatitis is still debated. There have been not many studies assessing the histologic outcome of initially mild chronic hepatitis C with sequential liver biopsies and most of them considered patients followed for a rather short time interval, usually of 2-5 years [10-13].
This period might be inadequate to assess the progressive or nonprogressive course of liver disease as the annual rate of fibrosis progression in hepatitis C is usually around 0.10-0.2 fibrosis units/year (METAVIR score) [14]. We report here the results obtained in 106 patients with initially mild chronic hepatitis C who underwent two liver biopsies 5-10 years (mean 7.8 years) apart without intervening antiviral treatment.
Discussion
Chronic hepatitis C represents a major health problem in Europe and in the US, being the main cause, either alone or with synergistic cofactors, of end-stage liver disease and hepatocellular carcinoma and the burden of disease is expected to increase further in the next decade [18,19].
Accordingly, there is the need to define which patients categories should be offered antiviral therapy, considering that the combination of PEG-IFN plus Ribavirin, the current standard of therapy for hepatitis C, produces a sustained virological response in more than 55-60% of treated patients [16,17]. These treatments have been shown to be more successful and better tolerated when given to patients with not too advanced liver disease [20], but their use in HCV carriers with no/minimal liver fibrosis has been questioned mainly because this has been assumed to be a benign, stable condition with minimal risk of progression to 'clinically significant' liver disease. Furthermore, current treatments are costly and associated with side-effects. The issue on whether liver fibrosis is or is not progressive in cases with hepatitis C and no/minimal fibrosis in the initial liver biopsy has been recently reassessed in studies where serial biopsies were taken in the absence of intervention with anti-viral therapy [10-13,21].
Most of these studies had a rather short (<5 years) time interval between biopsies and reported progression of liver disease only in a small minority of cases. Our present study represents the largest series of patients so far analysed, with the longest time interval between biopsies in the literature. All our patients had the final liver biopsy taken more than 5 years after the initial one and almost two-thirds of them showed fibrosis progression, development of advanced fibrosis/cirrhosis being observed in 27% including more than one-third of those with F1 (portal fibrosis) in the initial biopsy. These results clearly indicate that chronic hepatitis C is a progressive disease in many patients who initially present with no/minimal fibrosis and that progression to advanced fibrosis does occur in a significant number of cases within 5-10 years.
Comparing our findings with those reported previously by others, they appear congruent with the concept that the different time interval between biopsies is the obvious explanation for the different rates in disease progression and in the development of advanced fibrosis/cirrhosis observed. As previously reported by us [22] and recently confirmed by Ghany et al. [10], fibrosis progression in initially mild chronic hepatitis C is significantly influenced by patients age and by the ALT profile. Indeed, age at first biopsy directly correlated with fibrosis progression during follow-up confirming that HCV infection becomes more 'fibrogenic' with advancing host age [23]. Considering the ΔF/year observed in different age subgroups, our data indicate that patients >50 years can be predicted to develop cirrhosis from initially mild disease in half (15-20 years) the time interval estimated for those <35 years (time to cirrhosis: 30-40 years or more), mean ΔF/year being 0.20-0.25 in the former and <0.10 in the latter (see Fig. 2).
Our results fully confirmed that ALT levels are strongly associated with fibrosis progression in hepatitis C, particularly when several determinations rather than a single point are considered. Accordingly to our finding, each doubling in ALT levels is reflected in doubling ΔF/year, i.e. in the speed of fibrosis accumulation in the liver (see Fig. 4). Thus, ALT monitoring represent an easy, and extremely useful tool to assess prognosis of initially mild hepatitis C. Some progression of fibrosis was seen also in 29% of the patients with persistently normal ALT, however, we cannot exclude that some of these cases, that were evaluated only every 6-12 months, might have had abnormal ALT if checked more frequently. Other parameters that were independently associated with disease progression included liver steatosis and alcohol intake, indicating that these metabolic cofactors, already described to influence the course of chronic hepatitis C [24,25], are relevant also in the more specific setting of initially mild disease.
In conclusion, our results indicate that chronic hepatitis C presenting with no/minimal fibrosis in liver biopsy should not be considered as a benign, steadily nonprogressive form of hepatitis C. Considering the rates of progression we observed in our patients within 5-10 years, the effect on these of patient age, and the most unlikely prospective that better tolerated and less expensive treatments could become available during the next 5-10 years, it seems quite reasonable to conclude that all patients with histologically mild chronic hepatitis C should be considered for antiviral therapy when ALT are abnormal and there are no major contraindications to the use of PEG-IFN and Ribavirin.
Results
Rate of fibrosis progression in 106 HCV carriers with initially mild chronic hepatitis C As shown in Fig. 1, comparison between the initial and final liver biopsy demonstrated no changes in fibrosis stage in 42 cases (39.6% nonprogressors: 14 with F0 and 28 with F1) and variable degree of liver fibrosis progression in the remaining 64 patients (60.4% progressors: 13 with F0 and 51 with F1 in the initial biopsy). The ΔF was 1 stage in 27, 2 stages in 24 and 3 stages in 13 of these progressors. The probability of being nonprogressor or progressor did not differ significantly between cases with F0 or F1 in the initial biopsy (P = 0.201) and, among progressors, mean ΔF/year was 0.212 ± 0.66 in patients with F0 and 0.266 ± 0.91 in those with F1 (P = ns).
However a ΔF of 3 stages as well as progression to F3 or to F4 (cirrhosis) were observed only in patients with F1 in the initial biopsy. Overall, fibrosis progression was observed in 13 of 27 cases with F0 (48.1%, including 5 to F1 and 8 to F2) and in 51 of 79 with F1 (64.5%, including 22 to F2, 16 to F3 and 13 to F4). Thus, among those with F1 initially, 20% progressed to F3 and 16% to F4 (cirrhosis). Variables associated with liver fibrosis progression in initially mild chronic hepatitis C When nonprogressors and progressors were compared for baseline features and follow-up profiles no differences were seen as to gender and HCV genotype distribution (Table 2).
Mean age was significantly higher in patients with fibrosis progression during follow-up. The rates of fibrosis progression in different age subgroups are shown in Table 3. Accordingly, there was a highly statistically significant correlation between age at inclusion and ΔF during follow-up (P < 0.0001), as well as between age at inclusion and the annual fibrosis progression rate during follow-up (Fig. 2). Progressors showed a significantly higher prevalence of elevated ALT (79.6%vs 52.3%, P = 0.005) at baseline as well as of significant histological activity (A2 31.2%vs 2.3%, P = 0.0004) and of steatosis (46.8%vs 14.3%, P = 0.002) in the initial liver biopsy compared with nonprogressors (Table 2). As shown in Fig. 3, mean ΔF/t index was 0.092 ± 0.01 in patients without steatosis, 0.19 ± 0.02 in those with mild steatosis (<30%) and 0.28 ± 0.03 with severe (>30%) steatosis. Alcohol intake, either moderate (<40 g/day) or high (>40 g/day) was statistically (P = 0.008) more frequent among progressors compared with nonprogressors (Table 2). By multivariate analysis high alcohol intake (>40 g/day) and steatosis correlated independently with fibrosis progression (data not shown).
HBV co-infection, as well as baseline albumin, bilirubin, platelets and prothrombin time values did not differ between progressors and non progressors (data not shown). The ALT profile during follow-up was also found to have a significant association with fibrosis progression. Thirty-one patients had persistently normal ALT during the entire follow-up period and nine of them (29%) showed fibrosis progression (3 from F0 to F1, 5 from F1 to F2 and 1 from F1 to F3) compared with 55 of 75 (73.3%) of those with elevated ALT. Furthermore there was a clear relationship between mean ALT levels during follow-up and ΔF grade (Fig. 4) as well as between mean ALT levels during follow-up and the annual fibrosis rate (ΔF/year) in individual patients (Fig. 4).
Journal of Viral Hepatitis, 2006, 13, 297-302
ABSTRACT
Summary.
The natural history of chronic hepatitis C presenting with no/minimal liver fibrosis is uncertain with controversies on risk of progression and need for antiviral treatment. We studied rates and determinants of fibrosis progression in initially mild chronic hepatitis C.
One hundred and six patients (mean age 41.65 ± 12.83 years) with chronic hepatitis C virus infection and no/minimal fibrosis in the initial liver biopsy (F0/F1 by METAVIR score) were followed prospectively while untreated with repeated biopsy after 5 or more years (mean interval 7.8 ± 1.51 years).
Patients showing fibrosis progression were compared with nonprogressors for baseline and follow-up parameters. Sixty-four patients (60.4%) showed fibrosis progression including 13 of 27 (49%) with F0 and 51 of 79 (65%) with F1.
Progression to F3 or cirrhosis was seen in 36% of those with F1 initially.
Fibrosis progression (ΔF/year) was associated with age (P < 0.0001), baseline and follow-up alanine aminotransferase (ALT) (P = 0.005), histological activity (P = 0.004) and steatosis (P = 0.002)
in the initial biopsy and use of alcohol (P = 0.008).
Thus liver fibrosis progression occurs in two-thirds of patients with initially mild chronic hepatitis C within 5-10 years and advanced fibrosis/cirrhosis develops in one-third of those with F1 initially. Fibrosis is facilitated by older age and alcohol and associated with inflammatory activity and ALT levels. Antiviral therapy should be considered in mild chronic hepatitis C.
Introduction
Chronic infection with the hepatitis C virus (HCV) affects around 170-200 million persons worldwide and is the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma in most western countries [1,2]. In recent years, end-stage liver disease caused by HCV has become the major reason for liver transplantation in the US and in Europe. Many studies have been conducted during the past decade with the aim of defining the natural history and outcomes of chronic HCV infection and the major determinants of liver disease progression [3].
These studies have been difficult to perform due to: (a) the often silent course of chronic hepatitis C that remains asymptomatic for decades even in cases who eventually progress to cirrhosis; (b) the slow rate of progression in many cases, taking several decades for development of clinically meaningful complications; (c) the great heterogeneity of the disease as influenced by viral and host variables; (d) the availability of antiviral therapies of increasing efficacy that has determined a progressive reduction in the proportion of untreated patients among those diagnosed to have chronic hepatitis C.
The earliest studies conducted in patients with a well recognized source of infection (post-transfusional or otherwise parenterally contaminated cases) seemed to indicate that only a minority of HCV infected patients develop cirrhosis over a 15-25-year period, the proportion being particularly low when patients infected at younger age were considered [4-6]. Overall these studies demonstrated rather low morbidity and mortality due to hepatitis C during the first two to three decades after infection and tended to describe the disease as benign and nonprogressive.
On the contrary, other studies conducted in patients presenting with long-lasting and advanced liver disease, reported high rates of liver related complications and deaths during rather short follow-up periods [7]. Currently, one of the most clinically relevant while still controversial issues relates to the identification of patients at significant risk of progression among those who are diagnosed with chronic HCV infection while still asymptomatic and with histologically mild liver disease.
Recent studies indicate that this condition is frequently observed in the general population [8] and is also one of the main presentation profiles of chronic HCV infection in clinical practice as these patients are referred for consultation after having been diagnosed at occasional testing for anti-HCV.
The current attitude is to take a liver biopsy in these cases and to assess prognosis on the basis of the fibrosis stage [2,9]. Patients with no (F0) or minimal fibrosis limited to the portal tracts (F1) are considered to have mild, nonprogressive liver disease while those with more advanced fibrosis (≥F2) are considered at significant risk of developing cirrhosis (F4).
Accordingly, antiviral therapy is usually strongly recommended in the latter cases while the indication to treat mild chronic hepatitis is still debated. There have been not many studies assessing the histologic outcome of initially mild chronic hepatitis C with sequential liver biopsies and most of them considered patients followed for a rather short time interval, usually of 2-5 years [10-13].
This period might be inadequate to assess the progressive or nonprogressive course of liver disease as the annual rate of fibrosis progression in hepatitis C is usually around 0.10-0.2 fibrosis units/year (METAVIR score) [14]. We report here the results obtained in 106 patients with initially mild chronic hepatitis C who underwent two liver biopsies 5-10 years (mean 7.8 years) apart without intervening antiviral treatment.
Discussion
Chronic hepatitis C represents a major health problem in Europe and in the US, being the main cause, either alone or with synergistic cofactors, of end-stage liver disease and hepatocellular carcinoma and the burden of disease is expected to increase further in the next decade [18,19].
Accordingly, there is the need to define which patients categories should be offered antiviral therapy, considering that the combination of PEG-IFN plus Ribavirin, the current standard of therapy for hepatitis C, produces a sustained virological response in more than 55-60% of treated patients [16,17]. These treatments have been shown to be more successful and better tolerated when given to patients with not too advanced liver disease [20], but their use in HCV carriers with no/minimal liver fibrosis has been questioned mainly because this has been assumed to be a benign, stable condition with minimal risk of progression to 'clinically significant' liver disease. Furthermore, current treatments are costly and associated with side-effects. The issue on whether liver fibrosis is or is not progressive in cases with hepatitis C and no/minimal fibrosis in the initial liver biopsy has been recently reassessed in studies where serial biopsies were taken in the absence of intervention with anti-viral therapy [10-13,21].
Most of these studies had a rather short (<5 years) time interval between biopsies and reported progression of liver disease only in a small minority of cases. Our present study represents the largest series of patients so far analysed, with the longest time interval between biopsies in the literature. All our patients had the final liver biopsy taken more than 5 years after the initial one and almost two-thirds of them showed fibrosis progression, development of advanced fibrosis/cirrhosis being observed in 27% including more than one-third of those with F1 (portal fibrosis) in the initial biopsy. These results clearly indicate that chronic hepatitis C is a progressive disease in many patients who initially present with no/minimal fibrosis and that progression to advanced fibrosis does occur in a significant number of cases within 5-10 years.
Comparing our findings with those reported previously by others, they appear congruent with the concept that the different time interval between biopsies is the obvious explanation for the different rates in disease progression and in the development of advanced fibrosis/cirrhosis observed. As previously reported by us [22] and recently confirmed by Ghany et al. [10], fibrosis progression in initially mild chronic hepatitis C is significantly influenced by patients age and by the ALT profile. Indeed, age at first biopsy directly correlated with fibrosis progression during follow-up confirming that HCV infection becomes more 'fibrogenic' with advancing host age [23]. Considering the ΔF/year observed in different age subgroups, our data indicate that patients >50 years can be predicted to develop cirrhosis from initially mild disease in half (15-20 years) the time interval estimated for those <35 years (time to cirrhosis: 30-40 years or more), mean ΔF/year being 0.20-0.25 in the former and <0.10 in the latter (see Fig. 2).
Our results fully confirmed that ALT levels are strongly associated with fibrosis progression in hepatitis C, particularly when several determinations rather than a single point are considered. Accordingly to our finding, each doubling in ALT levels is reflected in doubling ΔF/year, i.e. in the speed of fibrosis accumulation in the liver (see Fig. 4). Thus, ALT monitoring represent an easy, and extremely useful tool to assess prognosis of initially mild hepatitis C. Some progression of fibrosis was seen also in 29% of the patients with persistently normal ALT, however, we cannot exclude that some of these cases, that were evaluated only every 6-12 months, might have had abnormal ALT if checked more frequently. Other parameters that were independently associated with disease progression included liver steatosis and alcohol intake, indicating that these metabolic cofactors, already described to influence the course of chronic hepatitis C [24,25], are relevant also in the more specific setting of initially mild disease.
In conclusion, our results indicate that chronic hepatitis C presenting with no/minimal fibrosis in liver biopsy should not be considered as a benign, steadily nonprogressive form of hepatitis C. Considering the rates of progression we observed in our patients within 5-10 years, the effect on these of patient age, and the most unlikely prospective that better tolerated and less expensive treatments could become available during the next 5-10 years, it seems quite reasonable to conclude that all patients with histologically mild chronic hepatitis C should be considered for antiviral therapy when ALT are abnormal and there are no major contraindications to the use of PEG-IFN and Ribavirin.
Results
Rate of fibrosis progression in 106 HCV carriers with initially mild chronic hepatitis C As shown in Fig. 1, comparison between the initial and final liver biopsy demonstrated no changes in fibrosis stage in 42 cases (39.6% nonprogressors: 14 with F0 and 28 with F1) and variable degree of liver fibrosis progression in the remaining 64 patients (60.4% progressors: 13 with F0 and 51 with F1 in the initial biopsy). The ΔF was 1 stage in 27, 2 stages in 24 and 3 stages in 13 of these progressors. The probability of being nonprogressor or progressor did not differ significantly between cases with F0 or F1 in the initial biopsy (P = 0.201) and, among progressors, mean ΔF/year was 0.212 ± 0.66 in patients with F0 and 0.266 ± 0.91 in those with F1 (P = ns).
However a ΔF of 3 stages as well as progression to F3 or to F4 (cirrhosis) were observed only in patients with F1 in the initial biopsy. Overall, fibrosis progression was observed in 13 of 27 cases with F0 (48.1%, including 5 to F1 and 8 to F2) and in 51 of 79 with F1 (64.5%, including 22 to F2, 16 to F3 and 13 to F4). Thus, among those with F1 initially, 20% progressed to F3 and 16% to F4 (cirrhosis). Variables associated with liver fibrosis progression in initially mild chronic hepatitis C When nonprogressors and progressors were compared for baseline features and follow-up profiles no differences were seen as to gender and HCV genotype distribution (Table 2).
Mean age was significantly higher in patients with fibrosis progression during follow-up. The rates of fibrosis progression in different age subgroups are shown in Table 3. Accordingly, there was a highly statistically significant correlation between age at inclusion and ΔF during follow-up (P < 0.0001), as well as between age at inclusion and the annual fibrosis progression rate during follow-up (Fig. 2). Progressors showed a significantly higher prevalence of elevated ALT (79.6%vs 52.3%, P = 0.005) at baseline as well as of significant histological activity (A2 31.2%vs 2.3%, P = 0.0004) and of steatosis (46.8%vs 14.3%, P = 0.002) in the initial liver biopsy compared with nonprogressors (Table 2). As shown in Fig. 3, mean ΔF/t index was 0.092 ± 0.01 in patients without steatosis, 0.19 ± 0.02 in those with mild steatosis (<30%) and 0.28 ± 0.03 with severe (>30%) steatosis. Alcohol intake, either moderate (<40 g/day) or high (>40 g/day) was statistically (P = 0.008) more frequent among progressors compared with nonprogressors (Table 2). By multivariate analysis high alcohol intake (>40 g/day) and steatosis correlated independently with fibrosis progression (data not shown).
HBV co-infection, as well as baseline albumin, bilirubin, platelets and prothrombin time values did not differ between progressors and non progressors (data not shown). The ALT profile during follow-up was also found to have a significant association with fibrosis progression. Thirty-one patients had persistently normal ALT during the entire follow-up period and nine of them (29%) showed fibrosis progression (3 from F0 to F1, 5 from F1 to F2 and 1 from F1 to F3) compared with 55 of 75 (73.3%) of those with elevated ALT. Furthermore there was a clear relationship between mean ALT levels during follow-up and ΔF grade (Fig. 4) as well as between mean ALT levels during follow-up and the annual fibrosis rate (ΔF/year) in individual patients (Fig. 4).
Table 3 Fibrosis progression in age subgroups of patients with initially mild hepatitis C
Materials and methods
Patients
This study was conducted on a cohort of 106 patients with initially mild chronic hepatitis C, identified from a database of 586 cases of chronic HCV infection seen at the Department of Clinical and Experimental Medicine of the University of Padova.
The inclusion criteria were: (a) chronic HCV infection diagnosed on the basis of serum anti-HCV and HCV-RNA positivity confirmed in two separate tests 6 months apart; (b) histologic evidence of mild chronic hepatitis with a liver biopsy showing F0 or F1 fibrosis stage according to the METAVIR classification [15]; (c) a follow-up period of at least 48 months without antiviral therapy with at least one sequential liver biopsy taken after this period of no treatment. Baseline and follow-up liver biopsies had to be at least 1.5 cm of length with presence of at least seven portal tracts for allowing inclusion of the patient. Patients were included independently of age, alanine aminotransferase (ALT) profile and presence of cofactors, such as alcohol use, hepatitis B virus (HBV) infection or other co-morbidities.
Patients were included on the basis of these criteria, without any other selection. There were 63 additional patients with F0 or F1 seen during the same time interval who could not be included as they were lost to follow-up and did not undergo a second liver biopsy, although this was initially recommended to all of them. Their baseline demographic and laboratory and clinical characteristics were not different from those of the 106 included patients. Twenty-three additional patients were excluded because either the baseline or follow-up biopsy was inadequate for histological assessment because of insufficient size.
The main characteristics of the 106 patients at the time of inclusion are summarized in Table 1. There were 59 male and 47 female patients aged 18-68 years (mean 41.65 ± 12.83 years). Initial histology showed no fibrosis (F0) in 27 cases and portal fibrosis without septa (F1) in the remaining 79 cases, accompanied by variable degree of necroinflammatory changes. All 106 patients had been followed-up with periodical (every 6-12 months) clinical assessment and liver function tests evaluation, which included ALT, aspartate aminotransferase, GGT, bilirubin, protrombin time and haematology.
Most patients underwent periodical ultrasound examination of the liver every 12-18 months. Patients did not receive antiviral treatment with interferon or interferon plus ribavirin, according to the Italian National guidelines that required histological grading of at least F2 as indication for treatment. All patients underwent a second liver biopsy 5-10 years after the initial one (mean interval between liver biopsies being 7.8 ± 1.51 years) to reassess stage and progression of liver disease. This time interval for taking a second liver biopsy was based on published data on the fibrosis progression rates that can be expected in chronic hepatitis C and on the recommendations given in 1999 by the EASL Consensus Conference on hepatitis C [9].
This study describes the changes seen when paired liver biopsies obtained over this time interval were compared. Patients were then not considered further for this analysis as many of them (68 out of 106) received antiviral therapy after the second histological evaluation either because of evidence of disease progression and/or as the consequence of more permissive criteria for initiating treatment adopted when PEG-IFN plus ribavirin combination therapy became available [16,17].
Histological evaluation
Liver sections obtained from paired biopsies in each individual patient were stained with haematoxylin/eosin and a reticulin stain. They were then scored for activity and fibrosis using the METAVIR system [15] by a Pathologist (M.G.) who was unaware of clinical findings and of the sequential order by which biopsies had been taken. Liver steatosis in the initial biopsy was classified as absent (grade 0), moderate with <30% of hepatocytes affected (grade1) or severe with >30% of hepatocytes affected (grade 2).
Patients were classified as nonprogressors or progressors according to changes in liver fibrosis score between the initial and the final biopsy. Progressors were further listed according to the grade of fibrosis progression observed (ΔF = final - initial fibrosis grade). The annual fibrosis progression rate in each individual case was calculated as the ratio between ΔF and number of years elapsed between the two biopsies [14].
Statistical analysis To investigate the association between variables and fibrosis progression we compared progressor and nonprogressor patients and analysed data using Fischer's exact test for categorical variables and Student t-test for continuous variables. To estimate independent association between steatosis (grade 0-2), alcohol intake (grade 0 = no alcohol, grade 1 = ≤40 g/day, grade 2 = >40 g/day) and fibrosis progression we used logistic regression. Statistical analyses were performed using the Statistical Program for Social Sciences (SPSS V-12, SPSS Inc., Chicago, IL, USA).
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Patients
This study was conducted on a cohort of 106 patients with initially mild chronic hepatitis C, identified from a database of 586 cases of chronic HCV infection seen at the Department of Clinical and Experimental Medicine of the University of Padova.
The inclusion criteria were: (a) chronic HCV infection diagnosed on the basis of serum anti-HCV and HCV-RNA positivity confirmed in two separate tests 6 months apart; (b) histologic evidence of mild chronic hepatitis with a liver biopsy showing F0 or F1 fibrosis stage according to the METAVIR classification [15]; (c) a follow-up period of at least 48 months without antiviral therapy with at least one sequential liver biopsy taken after this period of no treatment. Baseline and follow-up liver biopsies had to be at least 1.5 cm of length with presence of at least seven portal tracts for allowing inclusion of the patient. Patients were included independently of age, alanine aminotransferase (ALT) profile and presence of cofactors, such as alcohol use, hepatitis B virus (HBV) infection or other co-morbidities.
Patients were included on the basis of these criteria, without any other selection. There were 63 additional patients with F0 or F1 seen during the same time interval who could not be included as they were lost to follow-up and did not undergo a second liver biopsy, although this was initially recommended to all of them. Their baseline demographic and laboratory and clinical characteristics were not different from those of the 106 included patients. Twenty-three additional patients were excluded because either the baseline or follow-up biopsy was inadequate for histological assessment because of insufficient size.
The main characteristics of the 106 patients at the time of inclusion are summarized in Table 1. There were 59 male and 47 female patients aged 18-68 years (mean 41.65 ± 12.83 years). Initial histology showed no fibrosis (F0) in 27 cases and portal fibrosis without septa (F1) in the remaining 79 cases, accompanied by variable degree of necroinflammatory changes. All 106 patients had been followed-up with periodical (every 6-12 months) clinical assessment and liver function tests evaluation, which included ALT, aspartate aminotransferase, GGT, bilirubin, protrombin time and haematology.
Most patients underwent periodical ultrasound examination of the liver every 12-18 months. Patients did not receive antiviral treatment with interferon or interferon plus ribavirin, according to the Italian National guidelines that required histological grading of at least F2 as indication for treatment. All patients underwent a second liver biopsy 5-10 years after the initial one (mean interval between liver biopsies being 7.8 ± 1.51 years) to reassess stage and progression of liver disease. This time interval for taking a second liver biopsy was based on published data on the fibrosis progression rates that can be expected in chronic hepatitis C and on the recommendations given in 1999 by the EASL Consensus Conference on hepatitis C [9].
This study describes the changes seen when paired liver biopsies obtained over this time interval were compared. Patients were then not considered further for this analysis as many of them (68 out of 106) received antiviral therapy after the second histological evaluation either because of evidence of disease progression and/or as the consequence of more permissive criteria for initiating treatment adopted when PEG-IFN plus ribavirin combination therapy became available [16,17].
Histological evaluation
Liver sections obtained from paired biopsies in each individual patient were stained with haematoxylin/eosin and a reticulin stain. They were then scored for activity and fibrosis using the METAVIR system [15] by a Pathologist (M.G.) who was unaware of clinical findings and of the sequential order by which biopsies had been taken. Liver steatosis in the initial biopsy was classified as absent (grade 0), moderate with <30% of hepatocytes affected (grade1) or severe with >30% of hepatocytes affected (grade 2).
Patients were classified as nonprogressors or progressors according to changes in liver fibrosis score between the initial and the final biopsy. Progressors were further listed according to the grade of fibrosis progression observed (ΔF = final - initial fibrosis grade). The annual fibrosis progression rate in each individual case was calculated as the ratio between ΔF and number of years elapsed between the two biopsies [14].
Statistical analysis To investigate the association between variables and fibrosis progression we compared progressor and nonprogressor patients and analysed data using Fischer's exact test for categorical variables and Student t-test for continuous variables. To estimate independent association between steatosis (grade 0-2), alcohol intake (grade 0 = no alcohol, grade 1 = ≤40 g/day, grade 2 = >40 g/day) and fibrosis progression we used logistic regression. Statistical analyses were performed using the Statistical Program for Social Sciences (SPSS V-12, SPSS Inc., Chicago, IL, USA).
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