Laboratory approach to acute and chronic hepatitis.

Hepatitis is a common disease worldwide. While the incidence of acute viral hepatitis has fallen by over 80% in the United States in the past decade, there has been increased recognition of chronic viral hepatitis. An estimated 1 million U.S. residents are chronically infected with hepatitis B virus (HBV), while at least 2.7 million have chronic hepatitis C virus (HCV) infection. Chronic hepatitis can progress to cirrhosis, the tenth leading cause of death in the United States, and to hepatocellular carcinoma, the fifth leading cause of cancer death worldwide. The number of persons with these two serious consequences of chronic hepatitis is expected to increase two- to threefold in the United States by 2030 before declining. While viral hepatitis is the most widely known form, a number of other etiologies can cause both acute and chronic hepatitis.

Hepatitis is a disease primarily defined by laboratory tests. The clinical laboratory is critical to recognition of chronic hepatitis, and is involved in diagnosis of many cases of acute hepatitis, as well. Laboratory tests are needed also in the differential diagnosis of the cause of most cases of hepatitis. Recent guidelines have suggested approaches to use relative to laboratory tests in hepatic disease. (1, 2) This article summarizes the laboratory features of both acute and chronic hepatitis, and the most common laboratory tests needed to establish the precise etiology of liver damage.

Defining acute hepatitis

Acute hepatitis is defined as an acute injury to the liver, manifested by release of liver cytoplasmic enzymes (particularly aspartate aminotransferase [AST] and alanine aminotransferase [ALT]). In a variable percentage of cases, the increase in enzymes is accompanied by symptoms such as fever, loss of appetite and abnormal bilirubin metabolism (jaundice [yellow coloration of the skin and eyes], dark urine and pale stools). Acute hepatitis is usually a self-limiting disease, but may cause severe liver injury leading to liver Failure (fulminant hepatic failure) and the need for liver transplantation to prevent death and, with some causes, may progress to chronic hepatitis.

In those with acute hepatitis severe enough to seek medical attention, laboratory tests allow classification into patterns of liver injury and help define the cause. (1) In the cytoplasm of hepatocytes (the target of injury in acute hepatitis), there is approximately one and a half to two times as much AST as ALT. In the early stages of acute hepatitis, serum AST is typically higher than ALT. The half-life of AST is about 18 hours, while that of ALT is about 48 hours; thus, after the first one to two days of illness, ALT is typically higher than AST.

Two main patterns of liver injury are seen in acute hepatitis. In some cases, the damage to the liver is direct; this is typical of hepatitis due to toxins (primarily acetaminophen) (3) or to shock (ischemic hepatitis or shock liver). (4, 5) With these causes, AST and ALT increase rapidly, often to extremely high values, accompanied by marked prolongation of prothrombin time. Peak abnormalities usually occur by 24 to 48 hours after onset of injury, and then rapidly fall toward normal (as expected from their half-life). In contrast, most acute hepatitis is due to an immune reaction against infectious agents or drugs that damages hepatocytes. With these causes, AST and ALT rise slowly, reach a plateau, and fall gradually after several weeks of elevation; prothrombin time is prolonged more than three seconds only with the most severe cases. A third pattern occurs in alcoholic hepatitis. Alcohol has both direct effects on the liver (impaired protein synthesis) and causes an immunologic response. Because alcohol increases AST release and inhibits ALT production, in alcoholic hepatitis, AST remains higher than ALT. A summary of the typical changes in different causes of acute hepatitis is shown in Table 1.

Because viral hepatitis remains an important cause of acute hepatitis, laboratory detection of viral markers and antibodies is critical to diagnose acute viral hepatitis. Hepatitis A virus (HAV), spread by the fecal-oral route, has a very short incubation period of two to four weeks and (in adults) commonly causes jaundice. Because many adults have previously been exposed to HAV, detection of total antibody to HAV cannot be used to diagnose acute infection; IgM anti-HAV is present for only three to six months after exposure and is considered diagnostic for acute HAV infection.

Acute HCV infection is usually clinically silent; at most, 10% to 30% of adults develop jaundice. After an incubation period of two to four weeks, HCV RNA becomes detectable in plasma, and rapidly increases to very high levels ([10.sup.7] to [10.sup.8] IU/mL). After another two to six weeks, ALT begins to increase; in about 50% to 70% of cases, anti-HCV is present at the time clinical hepatitis is diagnosed, but it remains negative for several weeks to a few months in the remaining 30% to 50% of cases. (6) In up to 50% of cases, HCV RNA is cleared during this acute infectious phase; (7) in some, anti-HCV never develops, while in another 30% antibody declines gradually over time and eventually becomes negative. (8) In 50% to 80% of cases, however, chronic infection with HCV develops. Once this chronic phase is established, HCV infection does not spontaneously resolve (although anti-viral treatment may he effective, as discussed later).

HBV remains the most common cause of acute viral hepatitis in the United States. HBV is spread sexually, by injection exposure, and sometimes from close contact with persons with HBV; a mother with HBV can infect newborn infants easily. Jaundice occurs in about one-third to one-half of adults with HBV, but almost never in children less than five. After an incubation period of one to two months, hepatitis B surface antigen (HBsAg) becomes detectable; acute hepatitis generally does not occur until one to two months later, accompanied by development of IgM antibodies to the hepatitis B core antigen (anti-HBc). As the infection wanes, HBsAg becomes undetectable in most cases, and anti-HBs develops over an average of four to six months. Individuals with negative HBsAg and positive anti-HBs have traditionally been classified as having "resolved" HBV infection, and are said to have "cleared" the virus. With more sensitive HBV DNA assays, however, it is clear that small amounts of virus remain in the liver and in the blood; (9) while posing no apparent risk to the person infected, HBV can be transmitted to others, especially if organ transplantation occurs. In some individuals, if HBsAg remains positive more than six months, it is termed chronic HBV infection.

Alcoholic hepatitis is less commonly seen than in the past. Laboratory findings differ from those of other forms of hepatitis. AST and ALT are only minimally increased, with peak AST usually <300 IU/L. In contrast to most other forms of hepatitis, AST typically is increased more than ALT, and the ratio of AST/ALT is often >2:1. (10) Increases in bilirubin and prothrombin time are often out of proportion to the change in the enzymes. Alcoholic hepatitis often looks, clinically, like an infectious disease: fever and increased neutrophil count are often present.

Acute hepatitis is now most commonly due to factors other than viral infection. Shock commonly damages the liver; regardless of the cause (bleeding, infection, cardiac problems). The laboratory picture is similar with all causes of shock: rapid, often marked rises in AST and ALT with rapid decrease, marked prolongation of prothrombin time, and no to minimal increase in bilirubin. If the patient survives the underlying shock, recovery of liver function is usually complete.

Toxins, such as overdoses of acetaminophen, are a less common cause of acute hepatitis but the most common cause of fulminant hepatic failure. The laboratory picture is similar to that seen in ischemic hepatitis. Drug reactions are an increasingly common cause of acute hepatitis. In many cases, a rash accompanies the liver injury, sometimes by joint of renal problems, and increased eosinophil count. The laboratory picture is similar to that of viral hepatitis, although increases in alkaline phosphatase are more commonly found with drug reactions; (11) viral markers are absent. Drug reactions most commonly occur shortly after starting a new drug, but sometimes occur after a person has been on a medication for several weeks to a few months. Discontinuation of the medication often leads to resolution of all abnormalities.

We usually start evaluation of a person with acute hepatitis by reviewing the routine laboratory tests, comparing the patterns to those seen in Table 1. If the pattern suggests ischemic or toxic hepatitis and the clinical picture is consistent, no further testing is needed. Although viral hepatitis has become less common, an acute hepatitis panel (approved by CMS) is typically done. The acute hepatitis panel consists of IgM anti-HAV, IgM anti-HBc, HBsAg, and anti-HCV. Because antibody may be negative in up to hall of cases of acute HCV at the time of presentation, we also do HCV RNA if the person has risk factors for HCV. If the AST:ALT ratio is >1 and the peak AST is below 10 times the upper reference limit, alcoholic hepatitis is likely. If the diagnosis has not yet been established, less common causes (especially drugs) should be considered.

Recognizing chronic hepatitis

In contrast to the more dramatic symptoms of acute hepatitis, chronic hepatitis is almost always a clinically unapparent disease. In some cases, chronic hepatitis is associated with fatigue or a mild feeling of not being well. Often, these symptoms become more apparent to the person once the diagnosis is made. Chronic hepatitis is, thus, almost always recognized because of laboratory tests. In the case of chronic viral hepatitis, the most common form, individuals are often recognized us being affected when blood is tested us part of blood donation, life or health insurance examinations, or other forms of routine testing. In all forms of chronic hepatitis, the disease may come to attention because of elevated ALT and/or AST activities in plasma. In most cases, increases are less than four times the upper reference limits, and often less than twice the upper reference limit. Other laboratory tests are virtually always normal in chronic hepatitis until complications develop. Particularly with chronic HCV infection, ALT levels often fluctuate between normal and increased, and it is not unusual for a person to have five to 10 normal ALT values separating elevated ones. In about 10% to 15% of cases of chronic hepatitis, ALT values are continually normal. In such cases, liver damage on biopsy lends to be very mild, although severe injury can occasionally be found.

With such minimal symptoms and laboratory findings, one might wonder why there is any concern about chronic hepatitis. While most persons with chronic hepatitis do not develop serious complications, about 20% to 30% go on to develop cirrhosis. Of those with cirrhosis, approximately 3% to 5% per year develop hepatocellular carcinoma. In the United States, deaths from hepatocellular carcinoma have almost doubled since 1980, and are expected to double again over the next 20 years; the need for liver transplantation is expected to increase at least threefold over the same period. (12)

Unfortunately, it is not possible to predict which patients will develop these late complications. Cirrhosis is the result of scarring (fibrosis) in the liver; patients with no fibrosis are therefore less likely to progress than those with severe fibrosis. The most reliable way to gauge the amount of fibrosis is by liver biopsy. Combinations of laboratory tests correlate with the presence of fibrosis, and can predict which persons do not have fibrosis with reasonable accuracy; unfortunately, they do not determine how much fibrosis is present. (13, 14) Commonly ordered laboratory tests, such us plasma enzymes, albumin, and prothrombin time, do not detect fibrosis until cirrhosis is well established. Thus, all patients with chronic hepatitis should be considered at risk of developing these serious complications.

In the United States, the most common causes of chronic hepatitis are chronic HBV and HCV infection and a metabolic disorder called nonalcoholic steatohepatitis (NASH); some 80% to 90% of cases are due to one of these three causes. Drugs, (15) autoimmune hepatitis (16) and hemochromatosis (17) make up most of the remaining cases; details on these three causes can be found in the references, and they will not be discussed further here.

HCV is the most common cause of chronic hepatitis in most of North America, Europe and Japan; about 3 million Americans have chronic HCV infection. (18) Most individuals with chronic HCV were infected during the period of 1960 to 1980 from injection drug use, or from 1970 to 1990 from blood transfusion. Chronic HCV is usually first suspected by finding anti-HCV, or by measuring anti-HCV after finding elevated ALT, followed by detection of HCV RNA in blood. Treatment of HCV with long-acting (pegylated) interferon and ribaviran successfully eradicates virus in over half of cases. Several factors affect likelihood of response, most importantly the strain (genotype) of HCV. There are six major genotypes of HCV; genotype 1 is the most common in most of the world, while genotypes 2 and 3 make up about 25 % of infections in North America. These latter two genotypes respond well to treatment, with cure of infection in 75% to 80% of cases with only 24 weeks of treatment; with other genotypes, 48 weeks of treatment has a success rate of about 40% to 45%. (19) Before treatment, it is critical to measure genotype and the viral load of HCV RNA. With genotypes other than 2 of 3, viral load is rechecked after 12 weeks of treatment; if the viral load has fallen by less than two logs ("early virologic response"), then treatment success is highly unlikely and treatment can be stopped. After treatment is completed, HCV RNA is rechecked six months following the completion of therapy; negative RNA is termed a "sustained virologic response," and indicates cure of infection in >99% of cases.

HBV is the most common cause of chronic hepatitis worldwide, but is becoming less common among U.S. natives due to immunization of infants and children. It is defined by persistence of HBsAg more than six months after onset of acute hepatitis. There are two major forms of chronic HBV. In the more severe form, the virus continues to replicate, causing high plasma viral load and ongoing damage to liver cells (manifested by increased ALT). In the chronic HBV carrier state, the viral DNA becomes integrated into the liver cell DNA, and viral load is either at a very low level or undetectable (nonreplicating form). For many years, the HBV e antigen was used as a way to distinguish these two forms; HBeAg was positive in the replicating form and negative in the nonreplicating form. Mutant strains of HBV that cannot produce e antigen represent about 10% to 15% of infections in North America, but over half of infections in Asia, Africa and southern Europe. (20) Even when the mutant was not initially present, it may develop during the course of infection. More and more physicians are using viral load measurements in addition to or instead of HBeAg.

Treatment for chronic HBV is usually reserved for those with replicating forms of infection; a viral load > 100,000 copies/mL is considered high enough to consider treatment. Currently, most patients are treated with lamivudine or adefovir dipivoxil, both agents that are also effective against HIV. In contrast to HIV infection, suppression of viral replication occurs in only about 30% to 40% of patients; those with very high viral loads or low ALT before treatment are less likely to respond. Once treatment is stopped, viral replication remains suppressed in about 30% to 40% of those who originally responded, and recurs in the remainder. With lamivudine, mutants resistant to the drug can develop; a rise in liver enzymes in a person with previously normal values on treatment, or return of HBV DNA typically indicate development of a mutant. To date, only rare cases of mutants resistant to adefovir have been identified.

Nonalcoholic fatty liver disease (NAFLD) is the general term used to describe accumulation of fat in the liver; the most severe form of NAFLD is NASH. (21) NAFLD is seen in most individuals with obesity and diabetes, and NASH occurs in about 10% of those with fatty liver. Recent studies have emphasized that the upper reference limit for ALT (and AST) is typically derived from "healthy" persons, a group that includes those that are overweight; the studies have recommended determining the reference limits for these enzymes using only individuals with normal body weight. (22) If this were done, we would likely classify more individuals as having NASH. There are no specific laboratory features that permit diagnosis of NASH, but it is often accompanied by increased lipids (especially triglycerides) and glucose intolerance, if not diabetes.

A laboratory approach to diagnosis in a person with chronic hepatitis usually starts with measurement of anti-HCV and HBsAg. If either is positive, it is advisable to confirm active infection by measuring HCV RNA or HBV DNA, respectively. If viral markers are absent, it is standard to do tests to look for less common causes of chronic hepatitis, such as hemochromatosis (increased serum iron and ferritin, high transferrin saturation or low unsaturated iron binding capacity) and autoimmune hepatitis (increased immunoglobulins, positive anti-nuclear antibody and/or anti-smooth muscle antibody). If all these are negative and the patient is overweight or diabetic, a liver biopsy can help to recognize NASH.

[GRAPHICS OMITTED]

Additional Reading

1. Supplement to Hepatology, November, 2002, on the NIH Consensus Statement on Hepatitis C.

2. Two guidelines on Hepatitis B have also been published by the American Association for the Study of Liver Diseases (Lok A, McMahon B: Chronic hepatitis B. Hepatology 2001;34:1225-1241) and the European Association for the Study of the Liver (Valla D, de Franchis R, Hadengue A, et al. EASL international consensus conference on hepatitis B. Consensus statement (short version). J Hepatol 2003; 38:533-540)

Table 1--Laboratory features in acute hepatitis

CAUSE                                  VIRAL            DRUG-INDUCED

Peak ALT                              8 to 40             3 to 40
(x upper reference limit)
AST:ALT ratio                           <1                   <1

Duration of increase (weeks)             5           <2 if drug stopped
Alkaline hosphatase                    <3 in              >3 in 50%
(x upper reference limit))          90% to 95%
Prothrombin time (sec                   <3                    <3
above upper reference limit)
Frequency of jaundice (%)            10 to 70              20 to 30
Viral serologies                     Positive              Negative

CAUSE                            TOXIC/ISCHEMIC          ALCOHOLIC

Peak ALT                        10 to 100 or more          3 to 5
(x upper reference limit)
AST:ALT ratio                    Transiently >1              >2
                                for first 2 days
Duration of increase (weeks)         1 to 2                3 to 5
Alkaline hosphatase                    <2                >3 in 25%
(x upper reference limit)
Prothrombin time (sec                  >4                    <3
above upper reference limit)
Frequency of jaundice (%)              <10                50 to 70
Viral serologies                    Negative              Negative

References

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(2.) Dufour D, Lott J, Nolte F, Gretch D, Koff R, Saeff L. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests. Clin Chem 2000;46:2027-2049.

(3.) Singer A, Carracio T, Mofenson H. The temporal profile of increased transaminase levels in patients with acetaminophen-induced liver dysfunction. Ann Emerg Med 1995;26:49-53.

(4.) Dufour D, Teot L. Laboratory identification of ischemic hepatitis (shock liver). Clin Chem 1998;34:1287.

(5.) Gitlin N, Serio K. Ischemic hepatitis: widening horizons. Am J Gastroenterol 1992;87:831-836.

(6.) Barrera J, Bruguera M, Ercilla M, et al. Persistent hepatitis C viremia after acute self-limiting posttransfusion hepatitis C. Hepatology 1995;21:639-644.

(7.) Kenny-Walsh E. Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. N Engl J Med 1999;340:1228-1233.

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(9.) Yuki N, Nagaoka T, Yamashiro M, et al. Long-term histologic and virologic outcomes of acute self-limited hepatitis B. Hepatology 2003;37:1172-1179.

(10.) Nanji A, French S, Mendenhall C. Serum aspartate aminotransferase to alanine aminotransferase ratio in human and experimental alcoholic liver disease: relationship to histologic changes. Enzyme 1989;41:112-115.

(11.) Farrell G. Drug-induced hepatic injury. J Gastroenterol Hepatol 1997;12:S242-250.

(12.) Davis G, Albright J, Cook S, Rosenberg D. Projecting future complications of chronic hepatitis C in the United States. Liver Transpl 2003;9:331-338.

(13.) Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou Y, Poynard T. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet 2001;357:1069-1075.

(14.) Poynard T, Imbert-Bismut F, Ratziu V, et al. Biochemical markers of liver fibrosis in patients infected by hepatitis C virus: longitudinal validation in a randomized trial. J Viral Hepat 2002;9:128-133.

(15.) Zimmerman H. Drug-induced liver disease. Clin Liver Dis 2000;4:73-96.

(16.) Czaja A, Freese D. Diagnosis and treatment of autoimmune hepatitis. Hepatology 2002;6:479-497.

(17.) Tavill A. Diagnosis and management of hemochromatosis. Hepatology 2001;33:1321-1328.

(18.) Alter M, Kruszon-Moran D, Nainan O, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999;341:556-562.

(19.) Manns M, McHutchison J, Gordon S, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001;358:958-965.

(20.) Francois G, Kew M, Van DP, Mphahlele M, Meheus A. Mutant hepatitis B viruses: a matter of academic interest only or a problem with far-reaching implications? Vaccine 2001;19:3799-3815.

(21.) Clark J, Brancati F, Diehl A, Nonalcoholic fatty liver disease. Gastroenterology 2002;122:1649-1657.

(22.) Prati D, Taioli E, Zanella A, et al. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med 2002;137:1-10.

D. Robert Dufour, MD, is chief, Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Washington, DC, and professor of pathology, George Washington University Medical Center.