- Email: [email protected]
Hepatitis B and hepatitis C
Clin Liver Dis 6 (2002) 317 – 334
Hepatitis B and hepatitis C Stephen A. Geller, MD Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA
Viral hepatitis has been recognized for more than seven centuries. Although this is one of the oldest known diseases, proof of its infectious nature was only obtained in the 1940s in a series of experiments demonstrating the transmissibility of the disease [1]. The understanding of viral and nonviral causes of hepatitis expanded dramatically in the years that followed, propelled by the further development and use of percutaneous needle biopsy, which allowed for detailed correlations of morphology with clinical behavior with increasingly sophisticated laboratory testing [2]. Among the many important discoveries were the recognition of the hepatitis B surface antigen (HBsAg) in 1977 by Blumberg et al [3] and the identification of the hepatitis C virus (HCV) by Choo et al in 1989 [4]. The reported cases of viral hepatitis have declined dramatically in the last three decades, with the greatest decline in the incidence of hepatitis A [5]. Hepatitis B reached a peak of approximately 11 cases per 100,000 in the mid1980s, declining to approximately 4 cases per 100,000 in 1999. The rate of nonA, non-B hepatitis was at its highest in 1993, 3 years after the introduction of the first serologic test for hepatitis C, with approximately 3 cases per 100,000, falling to fewer than 2 cases per 100,000 in 1999. Although the acute forms of hepatitis B and hepatitis C are less prevalent, chronic hepatitis B and C remain major health care issues [6]. There are more than 300 million hepatitis B virus (HBV) carriers in the world, of whom more than 250,000 die each year from hepatitis B –associated chronic liver disease [7,8]. More than 1 million individuals in the United States are chronically infected with HBV [9,10]. HCV is also a major world health problem, with 100 million people infected worldwide, and almost 4 million people infected in the United States [11]. Chronic HCV infection has become the leading indication for liver transplantation in the United States; recurrent HCV infection after
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transplantation is almost universal, with half of the recipients developing clinically overt disease caused by HCV [12]. Hepatitis D (HDV; formerly termed delta hepatitis), which depends on HBV to manifest, is also an important cause of acute and chronic liver disease [13]. Although the molecular biology aspects of hepatitis B, D, and C are distinctive, their clinical manifestations and, to a considerable degree, morphologic consequences are quite similar.
Clinical manifestations Acute viral hepatitis The onset of viral hepatitis may be abrupt or gradual. The most common symptoms are fatigue, lassitude, drowsiness, nausea, and anorexia with associated low-grade fever and, if vomiting is prolonged, dehydration. Vague abdominal discomfort is common and some patients experience right upper quadrant abdominal pain. Joint symptoms may be prominent, especially with acute HBV. The urine can be dark, and the skin and sclerae may be icteric. Slight hepatomegaly is common, and some patients also experience splenomegaly. Symptoms often abate when jaundice develops. In severe hepatitis, which is unusual, confusion or stupor may develop and may progress to coma. Poor prognostic signs include asterixis, marked peripheral edema, and ascites. Chronic viral hepatitis Clinical manifestations can be diverse in chronic viral hepatitis [7,8]. In some patients, the only evidence of chronic hepatitis is persistent elevation of aminotransferase levels; this can occur in completely asymptomatic individuals. Other patients may have severe and steadily progressive disease, sometimes with rapid development of fulminant hepatic failure. Fatigue and generalized malaise, with mild abdominal pain, are the most common manifestations. Stigmata of chronic liver disease may be lacking. In more advanced cases, there may be anorexia, jaundice, hepatomegaly, ascites, jaundice, palmar erythema, spider angiomas, and encephalopathy.
Laboratory findings Acute viral hepatitis Many patients are slightly anemic at the time of presentation, and there may be a relative lymphocytosis. Serum bilirubin values are usually less than 20 mg/dL, except when there is severe disease and/or associated hemolysis. Aminotransferase values rise 1 to 2 weeks before the onset of jaundice and then begin to fall. However, the degree of aminotransferase elevation does not always parallel the
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severity of the hepatitis. Alkaline phosphatase is generally only slightly increased, except when cholestasis is prominent. Serum g-globulin levels may be slightly raised or normal. When hepatitis is severe, albumin levels are depressed and prothrombin times increased. Chronic viral hepatitis Serum levels of alanine aminotransferase, or ALT, and aspartate aminotransferase, or AST, are almost always elevated, often with values greater than 400 IU/L [8]. Serum bilirubin values are usually within the reference range, except when there is progressive liver failure, at which time decline of hepatic synthetic function also may become apparent, with prolongation of prothrombin time and decrease of serum albumin levels. Unlike in chronic autoimmune hepatitis (AIH), g-globulin values are not elevated.
Hepatitis B Pathogenesis and molecular biology HBV is the human prototype of the Hepadnaviridae family of viruses [14]. Hepadnaviruses are small, predominantly hepatotropic, enveloped DNA viruses with similar virion structure. The three types of particles present in infected individuals are as follows: the Dane particle, a 42-nm double-shelled particle [15]; filamentous particles of varying length with a 22-nm diameter [16]; and spherical 22-nm particles [17]. The Dane particle consists of a lipoprotein envelope surrounding an inner 27-nm nucleocapsid, within which circular DNA is attached to viral polymerase. A protein kinase activator of host origin is also present. The filamentous and spherical particles lack nucleocapsid and genomic DNA and are noninfectious. The major (S) envelope protein, on the surface of all three particle forms, represents a common antigen, HBsAg. HBsAg blood concentration can be quite high and is used as a diagnostic marker. HBsAg carries group and subtype determinants that are unevenly distributed throughout the world. Other glycoproteins of the virion envelope are middle, or M, and large, or L, envelope proteins. Hepatitis B core antigen (HBcAg) is composed of a phosphorylated capsid, or C, protein with an icosahedral structure with subunits clustered as a dimer. The HBV genome is a circular, partially double-stranded, 3.2-kilobase DNA molecule having a highly compact coding structure, with every nucleotide in a coding region and more than half of the genome translated in more than one open reading frame (ORF) [18]. The S-ORF, including pre-S and S regions, codes for the viral surface proteins. The ORF precore codes for a hydrophobic peptide that directs the transplantation product to the endoplasmic reticulum, where further protein cleavage forms the third HBV antigen, HBeAg. HbeAg is a highly reliable clinical serum marker for the presence of infectious virion [19]. On a more basic level, HBeAg may serve as an immunoregulatory protein [20,21]. The
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P gene codes for the viral polymerase (pol), which overlaps the entire length of the S-ORF. The pol has three domains that catalyze genome synthesis (reverse transcriptase domain) [15]; degrade pregenomic RNA, facilitating replication (Rnase H domain) [16]; and affect encapsidation and initiate negative strand synthesis (terminal protein domain) [17]. The fourth ORF codes for HBX, a protein whose exact function is not known. HBX may have multiple effector pathways and may also counteract the increased proteolytic function of the infected cells, ensuring the reliability of the replication process. Hepadnaviral infection begins with the attachment of mature virions to host cell membranes, involving the pre-S domain and other factors. Entry of the virus results from fusion of the viral and host membranes and release of the complete virus, the nucleocapsid, into the cytoplasm. DNA replication occurs through an RNA intermediate using viral-encoded reverse transcriptase [14,22]. Although there is only one major serotype of HBV, HBsAg has five major subtype determinants: a, d, y, w, and r. All HBsAg-positive sera contain determinant a; d and y are mutually exclusive, as are w and r. Therefore, four subtype patterns are seen: adw, ayw, adr, and ayr, although ayr is quite rare. The subtypes have beenassociated with the geographic distributions of HBV, but do not seem to correlate with clinical course. Subtype adw is most common in the Western Hemisphere and Europe, and adr prevails in most of the Far East. HBV is not cytopathic for hepatocytes, and viral hepatitis is caused by the cellular immune response to HBV-infected liver cells [23 – 25]. Cytokines emanating principally from cytotoxic T lymphocytes, but also from helper T cells, natural killer cells, and natural killer T cells, inhibit HBV expression by activating liver cells to degrade viral RNA while also inhibiting HBV replication by preventing the assembly of or disrupting the nucleocapsid particles within which replication occurs. Cytotoxic T cells kill hepatocytes via Fas ligand and perforin-induced apoptosis. Epidemiology of HBV HBV is transmitted primarily from infected serum or blood products. HBsAg and HBV-DNA can be detected in a variety of body fluids and secretions, the importance of which is unknown. In homosexual men, transmission is most often by oral or genital contact or from asymptomatic bleeding lesions in the rectal mucosa. Transmission to the fetus occurs during pregnancy.
Hepatitis D HDV is the smallest known genome to infect humans [26], encoding just one protein, the hepatitis delta antigen (HDAg) [27,28]. HDV depends on HBV as a helper. The HBV envelope is composed of lipid and HbsAg, with a ribonucleoprotein core consisting of HBV genome RNA complexed with HDAg. At least three phylogenetically distinct genotypes exist, varying in geographic distribution and clinical manifestations [21,29 – 31]. Genotype 1, the most prevalent, found in
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North America, Europe, the South Pacific, Asia, and Africa, is associated with a broad spectrum of chronic disease. Genotype 2, found only in East Asia, is associated with a mild form of HDV disease. Genotype 3, found in northern South America, causes a severe hepatitis. Acute coinfection, generally a self-limiting disease, occurs in patients not previously infected with HBV. Almost all patients recover completely, but some progress to chronic HBV-HDV infection, with eventual cirrhosis. A few of these patients progress to chronic hepatitis with persistence of both viruses. HDV superinfection occurs in individuals previously infected with HBV. The course of infection is more rapid than in coinfection, always with severe hepatitis. In as many as 20% of cases, HDV superinfection progresses to chronic HDV. In approximately 70% of cases, there is chronic HBV-HDV and ultimate cirrhosis [13]. HDV, in liver transplant recipients with HBV and HCV coinfection, is associated with suppression of HCV replication and only mild inflammatory activity [32]. With better control of HBV, HDV infection is declining in incidence [28,33] Hepatitis C Pathogenesis and molecular biology HCV is a member of the Flaviviridae family of viruses. Falviviridae are small, enveloped viruses containing a positive-sense, single-stranded RNA genome. This family includes the viruses of yellow fever, dengue, bovine viral diarrhea, and swine fever. HCV is the only member of the genus Hepacivirus. HCV is genetically heterogeneous, with six major genotypes worldwide and more than 100 subtypes. The genomes of the most different HCV isolates differ by up to 35% [34 – 37]. In the United States, almost half of the isolates are genotypes 1a and 1b. Considerable genotype variation is found among different population groups. The HCV genotype also is a factor in response to therapies. For example, patients with genotype 1b, particularly prevalent in the United States, respond poorly to both interferon and combined interferon-ribavirin when compared with patients infected with genotype 2 or 3. HCV circulates as quasispecies that result from mutations accumulated over time and present at the time of infection. Such mutations probably enable HCV to replicate efficiently or resist immune mechanisms. Quasispecies complexities influence the outcome of acute hepatitis or the severity of persistent hepatitis. The response to therapy is also affected [37]. The genetic complexity has hampered vaccine development. HCV viral genomic RNA is associated with the capsid protein (C) to form the spherical, 30 nm nucleocapsid, which is surrounded by a lipid-containing envelope with two viral glycoproteins (E1, E2), derived from host membranes, to form the infectious virion. E1 and E2 proteins exhibit a high degree of genetic heterogeneity. The highly variable N-terminus of the E2 protein has been designated the hypervariable region 1 (HVR-1). HVR-2 is found within the
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envelope E2 protein of genotype 1b. HVR-1 may represent a neutralization epitope for humoral immunity. Indeed, the presentation of antibodies against HVR-1 can be followed by the emergence of new variants, against which antibodies may not be identifiable. The core protein of HCV is highly conserved with several B-cell epitopes that contributed to the development of practical serologic tests for HCV. Core protein C-terminus has a hydrophobic region that aids transport to the endoplasmic reticulum and membrane-dependent core processing. The translocation of core into the nucleus has been suggested as a mechanism of cell transformation [16]. The mechanism by which HCV enters and infects cells is not known. Most likely, E1 and E2 are involved in receptor binding with subsequent fusion with the host cell. HCV may form lipoprotein complexes and enter cells by endocytosis via lipoprotein receptors [16]. The specific role of lymphocytes remains to be elucidated [38]. Epidemiology of hepatitis C HCV is transmitted in a variety of ways, including transfusion of blood products [39,40]. HCV is food-borne and endemic in most countries [41]. Transfusion-related transmission is still common throughout the world, although declining rapidly in the United States. Transmission also occurs with unsafe injections and can also occur with hemodialysis. Occupational exposure is uncommon, and the prevalence of HCV infection among health care workers is no greater than that found in the general population. The role of sexual activity remains controversial. In a small group of people, a specific route of infection cannot be identified.
Liver biopsy findings Acute viral hepatitis The histopathologic features of the viral hepatitides are similar, regardless of cause. In acute hepatitis with focal necrosis, morphologic changes are distinctive, although not pathognomonic. Portal tracts usually show varying chronic inflammatory cell infiltrate, but lobular changes are dominant. At low magnification, the biopsy of the liver in a patient with acute hepatitis has a disordered or ‘‘dirty’’ appearance (Fig. 1). In fully developed acute hepatitis, changes are predominantly seen in zone 3 of the acinus (centrolobular and perivenular), and to a lesser extent in zone 1 (portal and periportal). Zone 3 hepatocytes show ballooning and degenerative change, with apoptotic cell necrosis and acidophilic (Councilman-like) body formation (Fig. 2). Ballooned hepatocytes have distended, pale, eosinophilic, and finely granular cytoplasm. Nuclei are hyperchromatic and vary in size. Acidophilic bodies result from apoptosis genetically programmed cell death, at least partly controlled by
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Fig. 1. Acute hepatitis B. The liver has a disordered appearance, partially obscuring the usual architecture, with irregularly distributed lymphocytes, swollen hepatocytes, and scattered acidophilic bodies.
the bcl-2 oncogene. Acidophilic bodies can be numerous, and are recognized as refractile, deeply eosinophilic bodies, sometimes with fragments of the nuclear chromatin of the dying hepatocyte. They are commonly surrounded by a mostly T-lymphocyte infiltrate, implicating a role for immunologically mediated cell injury and death [42,43]. Acidophilic bodies can also be seen without lymphocytes (‘‘naked acidophilic bodies’’ or ‘‘tombstones’’). Histiocytes and occasional plasma cells are also seen [43,44]. In contrast to hepatitis B and C, acute hepatitis A may be rich in plasma cells, sometimes mimicking AIH [15]. Reactive Kupffer cells phagocytose debris and are also prominent, whatever the cause.
Fig. 2. Acute hepatitis B, showing two acidophilic bodies (arrows).
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After cell death and removal of cell products, areas of ‘‘drop-out’’ are seen in which the liver plate structure is temporarily maintained despite the loss of one or more hepatocytes; this is easily shown with reticulin stain. Kupffer cells contain periodic acid-Schiff (PAS)-positive material, best appreciated after diastase digestion (dPAS) to remove liver cell glycogen. PAS-positive Kupffer cells and small aggregates of PAS-positive macrophages in portal tracts, although entirely nonspecific, can be indicators of recent hepatitis when cell necrosis and inflammation are not obvious. The loss of many liver cells causes extensive architectural disarray of liver cell plates, with a range of histologic patterns, including confluent, bridging, or submassive and massive necrosis; these changes are also well highlighted with reticulin stain. Acute viral hepatitis with confluent necrosis is the term used when focal necroses become numerous, coalesce, and cause cell loss in large areas of liver parenchyma. When confluent necrosis involves multiple acini, it takes the form of acute hepatitis with multiacinar (panacinar, submassive, massive) necrosis. Bridging necrosis often involves all three acinar zones, forming necroinflammatory connections between portal and perivenular areas (portal-central or portalto-portal bridging necrosis; Fig. 3). The pathogenetic mechanisms of these two types of bridging necrosis may be different. Furthermore, perhaps because of vascular shunting, portocentral bridging necrosis has a poorer prognosis in both acute and chronic hepatitis. In acute hepatitis, this pattern is associated with a high risk for progression into chronic hepatitis and ultimately cirrhosis. Some patients, particularly those with extensive bridging necrosis, die within weeks to months after the onset of hepatitis with subfulminant liver failure. Complete recovery, however, can also occur. When there is necrosis of many adjacent liver cells, the underlying reticulin network undergoes ‘‘collapse’’ (Fig. 4). Collapse, when shown with hematoxylineosin –stained tissue, is often indistinguishable from newly formed fibrous septa.
Fig. 3. Acute hepatitis B, with bridging necrosis and inflammation.
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Fig. 4. Acute hepatitis B, with an area of collapse (reticulin).
Reticulin stain highlights the compressed network of closely arranged reticulin fibers. True cirrhotic septa include elastic fibers, demonstrated with Victoria blue or orcein methods, as well as with other stains. In recent collapse, there are no elastic fibers [45]. Multinuclear, giant cells, formed by the fusion of hepatocytes, are shown in Fig. 5. They occur in children (giant-cell hepatitis), and are associated with a variety of infectious and noninfectious etiologies; giant cells are also seen in acute viral hepatitis in adults, including HCV [46]. Portal tracts are generally mildly expanded, with an infiltrate of lymphocytes, histiocytes, some plasma cells, and, sometimes, a few polymorphonuclear
Fig. 5. Acute hepatitis C, showing multinucleation of hepatocytes.
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leukocytes (PMNs) and eosinophils. If PMNs and/or eosinophils predominate, causes other than viral should be considered, especially drugs or toxins. Interface hepatitis (piecemeal necrosis) is characterized by inflammatory cells spilling into the limiting plate hepatocytes. Interface hepatitis is associated with a greater potential for progression and ultimate development of chronic hepatitis but is not as important as confluent necrosis. Viral hepatitis severity and progression is related more to the cause of the hepatitis than the inflammation pattern. Acute hepatitis A and E can have severe liver inflammation, including prominent interface hepatitis, but neither progresses to chronic liver disease. Hepatitis C often has a relatively mild inflammatory cell component, but has a high propensity for chronicity [14]. Cholestasis also may be seen, but it is generally mild and confined to canaliculi. True cholestatic variants of hepatitis can occur and may be prolonged.
Chronic hepatitis Chronic hepatitis B Chronic hepatitis B shows a full spectrum of histologic changes, including those described as ‘‘chronic persistent’’ and ‘‘chronic active hepatitis,’’ terms no longer useful, with progression to cirrhosis [47]. With active virus replication, portal and/or lobular hepatitis of varying degree is always present [48]. HBcAg can be immunohistochemically demonstrated in hepatocyte nuclei (Fig. 6) [49]. The 25-nm core particles of HBcAg correspond to the inner Dane particle compartment [50]. With increasing inflammation, core antigen may become obvious in the cytoplasm [51]. The e antigen, characteristic of acute hepatitis, can
Fig. 6. Recurrent hepatitis B, after orthotopic liver transplantation, showing abundant HBcAg (immunoperoxidase). (See also Color Plate 1.)
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also be seen. The e antigen in the serum is associated with HBV replication, correlating with serum HBV-DNA and DNA polymerase, and hepatocytes HBcAg. The e antigen expresses in both nucleus and cytoplasm. HbsAg can also accumulate in the replicative stage, demonstrable histochemically, with Victoria blue or orcein (Shikata), or immunohistochemically. Portal hepatitis (‘‘chronic persistent’’) is generally seen during the phase of low virus replication. Portal or lobular hepatitis may reoccur with either virus reactivation or superinfection with another virus, such as HDV [51]. In HBsAg carriers, in the absence of active replication, the liver biopsy may lack inflammation or there may be mild portal hepatitis. Typically, ground-glass hepatocytes containing HBsAg may be seen (Fig. 7). Ground-glass cells are slightly enlarged with increased, finely granular, pale, and eosinophilic cytoplasm. With formalin fixation, a clear halo separates the granular cytoplasm from the nuclear membrane. Ground-glass cells may be widely dispersed, may occur in nodular clusters, or may be so sparse as to be almost unrecognizable with both hematoxylin-eosin and Victoria blue (Fig. 7) or orcein, and identified only with immunohistochemical methods. When cirrhosis develops, the inflammatory component may disappear, although ground-glass cells may persist. Chronic hepatitis D When HBV and HDV infection occur at the same time, the patient may have an acute hepatitis, which is sometimes fulminant. Replication of HDV may occur without HBV replication, however, and HDV infection may occur in HBsAg carriers who do not have active HBV infection. Chronic HDV hepatitis occurs in this setting [13]. However, HBV must be present before HDV causes recognizable cell injury and disease [27].
Fig. 7. Chronic hepatitis B, carrier state, showing (left) ground-glass hepatocytes and (right)HBsAg with Victoria blue stain. (See also Color Plate 2.)
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There are no specific clinical features that identify patients with chronic HDV [52]. The diagnosis should be considered in any HbsAg-positive patients, but especially in cases of HBV chronic hepatitis that rapidly progress to cirrhosis, in known chronic HBV patients whose clinical course suddenly worsens, and in patients from areas (Brazil, Venezuela, southern Italy, the Middle East) where HDV is prevalent. HDV occurs only sporadically in North America and affects mostly high-risk groups, such as hemodialysis patients, intravenous drug users, hemophiliacs, homosexuals, and prison inmates. HDV may also rarely be transmitted in blood or blood products used for transfusion, when screening tests fail to detect HBsAg. When infection with HDV is accompanied by HBV, there is always significant histologic injury [53 – 55]. Sometimes only portal hepatitis is seen. Generally, the biopsy shows more severe inflammation and severe liver disease, progressing to either liver failure or cirrhosis. There are no specific histologic changes that differentiate HDV from HBV. It has been suggested that a fatnegative, foamy, cytoplasmic degeneration is indicative of HDV [54], but this has not proven to be specific. Chronic hepatitis C The cloning and sequencing of HCV, the agent responsible for most of the cases of non-A, non-B hepatitis, as well as many cases of ‘‘cryptogenic’’ cirrhosis, and the subsequent development of a clinically useful assay [4,56] were major triumphs of the new discipline of molecular biology. Using polymerase chain reaction, HCV is recoverable from virtually all cases of chronic HCV, including cirrhosis, but not from AIH or cryptogenic cirrhosis [57,58] Chronic HCV infection occurs all over the world, and is more common than HBV in North America [9,11]. As many as 40% to 50% of patients will progress to chronic hepatitis, and as many as 20% of these individuals will become cirrhotic [7,16,59 – 61]. A series of articles has helped define the key histologic features of chronic HCV [50,59,62 –74]. The key features of chronic HCV are as follows: (1) marked and patchy expansion of portal tracts by predominantly lymphocytic infiltrate with minimal spilling over (piecemeal necrosis) into adjacent lobules, often with a well-defined lymphoid aggregate, including true follicle formation with a welldefined germinal center; (2) varying degrees of bile duct damage and focal bile duct loss [66]; (3) varying degrees of steatosis, including micro- and macrovesicular fat; and (4) sinusoidal cell hyperplasia. Steatosis may be associated with accelerated disease [16]. The low-magnification appearance of the liver biopsy is characteristic: patchy enlargement of portal tracts, often in a distinctly globular form (Fig. 8), with partially or well-developed lymphoid follicles, minimal piecemeal necrosis, prominent sinusoidal cells, and little to moderate steatosis. Less commonly seen features include (1) slight lobular necrosis, (2) liver cell dysplasia, (3) multinucleation, and (4) accumulations of Mallory-like material in
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Fig. 8. Chronic hepatitis C, showing globular expansion of a portal tract with minimal interface hepatitis and mild lobular hepatitis. There is also minimal macrovesicular steatosis.
hepatocytes. Liver cell regeneration is generally not appreciated in routine sections in early stages, but can often be demonstrated immunohistochemically. The previously described features are not pathognomonic and have, individually and in groups, been recognized in other forms of chronic hepatitis, including HBV; however, they are most often seen in HCV. The constellation of the first set of features described, not uncommonly seen as a group, is virtually diagnostic. Differential diagnosis includes other forms of chronic hepatitis, including AIH. Generally, true lymphoid follicles are not seen in those conditions, and other features, such as piecemeal necrosis, severe lobular necrosis and inflammation, and parenchymal collapse, are prominent. Immunohistochemical and molecular methods, including in situ polymerase chain reaction (PCR), for identifying HCV in tissue are not yet widely available, although they have been evaluated in the research setting. The ‘‘gold standard’’ for diagnosis has been the identification of viral RNA in tissue homogenates using PCR [57,75]. Laser-capture microdissection with subsequent PCR has shown HCV to concentrate in zone 1 [76]. The chronic hepatitis report: grading and staging The terms for chronic hepatitis developed in the 1970s and 1980s to express prognostic judgments about liver biopsy findings (chronic active hepatitis [CAH], chronic persistent hepatitis [CPH], chronic lobular hepatitis [CLH]) are no longer useful or acceptable, particularly because they do not reliably represent the biologic behavior of the disease [77]. For example, the high virus replicative stage of beginning chronic HBV, when HBeAg and high levels of HBV-DNA are demonstrable in serum, may show great variation in histologic appearance; there may be changes of mild CAH, CLH, or even CPH [78 –81]. This is followed by the stage of seroconversion, with anti-HBe appearing in the serum; severe CAH
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with considerable lobular activity is generally seen, although there may be either CAH or CPH with either virus reactivation or superinfection with another virus, such as HDV [53]. Finally, if cirrhosis does develop, the inflammatory component may disappear entirely. Chronic hepatitis biopsies should be reported in a way to (1) convey the diagnosis of chronic hepatitis itself; (2) portray the degree of inflammation; (3) describe the extent, if any, of fibrosis; and (4) indicate, if possible, the cause. A variety of numerical schemes have been developed over the years. The best known of these is the Knodell approach [82] that, like the others, suffers from a lack of reproducibility and a lack of widespread acceptability [77,79,83]. If a scoring system is used, instead of simply using English-language terms, the report should identify the system and provide the scale of scoring. English language, without numerical scoring, remains acceptable and is still widely used by hepatopathologists [77]. The reproducibility of numerical systems increases, of course, with the numbers of cases studied and can be best used in large centers with many cases. The principal purpose of biopsy grading and staging is to document the specific state of potentially progressive disorders, such as the chronic hepatitides, and also to help hepatologists determine whether antiviral therapies should be applied. A mutually understandable reporting format is the key to achieving these goals. Recurrence after liver transplantation Hepatitis B and C will recur in the transplanted liver, if appropriate preventive therapy is not applied. The histologic features of recurrent disease are similar to those of naturally occurring disease, although in the early stages of recurrent hepatitis C, differentiation from early acute allograft rejection may be problematic [70]. The topic of liver transplantation is discussed in another article in this issue.
Hepatocarcinogenesis Hepatocellular carcinoma (HCC) accounts for as many as 500,000 deaths per year worldwide [84] and is discussed in detail elsewhere in this issue. However, there is widespread agreement that HBV plays a major role in the development of HCC, with the risk of developing HCC 20 times greater in HBsAg-positive individuals than in seronegative controls [85 –87]. Strong epidemiologic evidence and animal models [88] confirm this. In addition, HBV-DNA has been found integrated in both tumor cells and adjacent liver tissue in the vast majority of HCC patients with demonstrable HBsAg, as well as in a few of those who are HBsAg-negative [89,90]. There is currently no conclusive evidence that HBV is directly oncogenic. Instead, it is thought that chronic HBV directly induces HCC by either activating cellular oncogenes or by inactivating tumor suppressor genes, or indirectly as a consequence of chronic liver injury, inflammation, and regeneration. Integration of HBV-DNA can also induce carcinogenesis.
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The risk of developing HCC is approximately 25 times greater for HCV carriers compared with noncarriers [86,91,92]. In hepatitis C, it has been shown that transgenic mice expressing the HCV core protein develop HCC [93], but the precise basis for HCC in chronic HCV infection is not known.
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[75] Barrett S, Kieran N, Ryan E, O’Keane C, Crowe J. Intrahepatic hepatitis C viral status of serum polymerase chain reaction-negative individuals with histological changes on liver biopsy. Hepatology 2001;33:1496 – 502. [76] Khanafshar E, Fong T-L, Chan R-C, Nichols WS, Geller SA. Hepatitis C virus localizes to zone 1 in chronic hepatitis C: a laser microdissection study. Hepatology 2001;34:431a. [77] Brunt EM. Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond. Hepatology 2000;31:241 – 6. [78] Hubscher SG. Histological grading and staging in chronic hepatitis: clinical applications and problems. J Hepatol 1998;29:1015 – 22. [79] Hunt N, Fleming K. Reproducibility of liver biopsy grading and staging. Liver 1999;19:169 – 70. [80] Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13: 372 – 4. [81] Scheuer PJ. Scoring of liver biopsies: are we doing it right? Eur J Gastroenterol Hepatol 1996; 8:1141 – 3. [82] Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;1:431 – 5. [83] Westin J, Lagging LM, Wejstal R, Norkrans G, Dhillon AP. Interobserver study of liver histopathology using the Ishak score in patients with chronic hepatitis C virus infection. Liver 1999; 19:183 – 7. [84] Murray CJ, Lopez AD. Mortality by cause for eight regions of the world: global burden of disease study. Lancet 1997;349:1269 – 76. [85] Bralet MP, Regimbeau JM, Pineau P, et al. Hepatocellular carcinoma occurring in nonfibrotic liver: epidemiologic and histopathologic analysis of 80 French cases. Hepatology 2000;32: 200 – 4. [86] Donato MF, Boffetta P, Puoti M. A meta-analysis of epidemiological studies on the combine effect of hepatitis B and C virus infections in causing hepatocellular carcinoma. Int J Cancer 1998;75:347 – 54. [87] Kawai H, Suda T, Aoyagi Y, et al. Quantitative evaluation of genomic instability as a possible predictor for development of hepatocellular carcinoma: comparison of loss of heterozygosity and replication error. Hepatology 2000;31:1246 – 50. [88] Popper H, Roth L, Purcell RH, Tennant BC, Gerin JL. Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc Natl Acad Sci USA 1987;84:866 – 70. [89] Koshy R, Maupas P, Muller R, Hofschneider PH. Detection of hepatitis B virus-specific DNA in the genome of human hepatocellular carcinoma and liver cirrhosis tissues. J Gen Virol 1981;57: 95 – 102. [90] Lok ASF, Ma OCK. Hepatitis B virus replication in Chinese patients with hepatocellular carcinoma. Hepatology 1990;12:582 – 8. [91] Aizawa Y, Shibamoto Y, Takagi I, Zeniya M, Toda G. Analysis of factors affecting the appearance of hepatocellular carcinoma in patients with chronic hepatitis C. A long term follow-up study after histologic diagnosis. Cancer 2000;89:53 – 9. [92] Bruno S, Silini E, Crosignani A, et al. Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study. Hepatology 1997;25:754 – 8. [93] Moriya K, Fujie H, Shintani Y, et al. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 1998;4:1065 – 7.
Hepatitis B and hepatitis C Stephen A. Geller, MD Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA
Viral hepatitis has been recognized for more than seven centuries. Although this is one of the oldest known diseases, proof of its infectious nature was only obtained in the 1940s in a series of experiments demonstrating the transmissibility of the disease [1]. The understanding of viral and nonviral causes of hepatitis expanded dramatically in the years that followed, propelled by the further development and use of percutaneous needle biopsy, which allowed for detailed correlations of morphology with clinical behavior with increasingly sophisticated laboratory testing [2]. Among the many important discoveries were the recognition of the hepatitis B surface antigen (HBsAg) in 1977 by Blumberg et al [3] and the identification of the hepatitis C virus (HCV) by Choo et al in 1989 [4]. The reported cases of viral hepatitis have declined dramatically in the last three decades, with the greatest decline in the incidence of hepatitis A [5]. Hepatitis B reached a peak of approximately 11 cases per 100,000 in the mid1980s, declining to approximately 4 cases per 100,000 in 1999. The rate of nonA, non-B hepatitis was at its highest in 1993, 3 years after the introduction of the first serologic test for hepatitis C, with approximately 3 cases per 100,000, falling to fewer than 2 cases per 100,000 in 1999. Although the acute forms of hepatitis B and hepatitis C are less prevalent, chronic hepatitis B and C remain major health care issues [6]. There are more than 300 million hepatitis B virus (HBV) carriers in the world, of whom more than 250,000 die each year from hepatitis B –associated chronic liver disease [7,8]. More than 1 million individuals in the United States are chronically infected with HBV [9,10]. HCV is also a major world health problem, with 100 million people infected worldwide, and almost 4 million people infected in the United States [11]. Chronic HCV infection has become the leading indication for liver transplantation in the United States; recurrent HCV infection after
E-mail address: [email protected] (S.A. Geller). 1089-3261/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 3 2 6 1 ( 0 2 ) 0 0 0 0 8 - 9
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transplantation is almost universal, with half of the recipients developing clinically overt disease caused by HCV [12]. Hepatitis D (HDV; formerly termed delta hepatitis), which depends on HBV to manifest, is also an important cause of acute and chronic liver disease [13]. Although the molecular biology aspects of hepatitis B, D, and C are distinctive, their clinical manifestations and, to a considerable degree, morphologic consequences are quite similar.
Clinical manifestations Acute viral hepatitis The onset of viral hepatitis may be abrupt or gradual. The most common symptoms are fatigue, lassitude, drowsiness, nausea, and anorexia with associated low-grade fever and, if vomiting is prolonged, dehydration. Vague abdominal discomfort is common and some patients experience right upper quadrant abdominal pain. Joint symptoms may be prominent, especially with acute HBV. The urine can be dark, and the skin and sclerae may be icteric. Slight hepatomegaly is common, and some patients also experience splenomegaly. Symptoms often abate when jaundice develops. In severe hepatitis, which is unusual, confusion or stupor may develop and may progress to coma. Poor prognostic signs include asterixis, marked peripheral edema, and ascites. Chronic viral hepatitis Clinical manifestations can be diverse in chronic viral hepatitis [7,8]. In some patients, the only evidence of chronic hepatitis is persistent elevation of aminotransferase levels; this can occur in completely asymptomatic individuals. Other patients may have severe and steadily progressive disease, sometimes with rapid development of fulminant hepatic failure. Fatigue and generalized malaise, with mild abdominal pain, are the most common manifestations. Stigmata of chronic liver disease may be lacking. In more advanced cases, there may be anorexia, jaundice, hepatomegaly, ascites, jaundice, palmar erythema, spider angiomas, and encephalopathy.
Laboratory findings Acute viral hepatitis Many patients are slightly anemic at the time of presentation, and there may be a relative lymphocytosis. Serum bilirubin values are usually less than 20 mg/dL, except when there is severe disease and/or associated hemolysis. Aminotransferase values rise 1 to 2 weeks before the onset of jaundice and then begin to fall. However, the degree of aminotransferase elevation does not always parallel the
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severity of the hepatitis. Alkaline phosphatase is generally only slightly increased, except when cholestasis is prominent. Serum g-globulin levels may be slightly raised or normal. When hepatitis is severe, albumin levels are depressed and prothrombin times increased. Chronic viral hepatitis Serum levels of alanine aminotransferase, or ALT, and aspartate aminotransferase, or AST, are almost always elevated, often with values greater than 400 IU/L [8]. Serum bilirubin values are usually within the reference range, except when there is progressive liver failure, at which time decline of hepatic synthetic function also may become apparent, with prolongation of prothrombin time and decrease of serum albumin levels. Unlike in chronic autoimmune hepatitis (AIH), g-globulin values are not elevated.
Hepatitis B Pathogenesis and molecular biology HBV is the human prototype of the Hepadnaviridae family of viruses [14]. Hepadnaviruses are small, predominantly hepatotropic, enveloped DNA viruses with similar virion structure. The three types of particles present in infected individuals are as follows: the Dane particle, a 42-nm double-shelled particle [15]; filamentous particles of varying length with a 22-nm diameter [16]; and spherical 22-nm particles [17]. The Dane particle consists of a lipoprotein envelope surrounding an inner 27-nm nucleocapsid, within which circular DNA is attached to viral polymerase. A protein kinase activator of host origin is also present. The filamentous and spherical particles lack nucleocapsid and genomic DNA and are noninfectious. The major (S) envelope protein, on the surface of all three particle forms, represents a common antigen, HBsAg. HBsAg blood concentration can be quite high and is used as a diagnostic marker. HBsAg carries group and subtype determinants that are unevenly distributed throughout the world. Other glycoproteins of the virion envelope are middle, or M, and large, or L, envelope proteins. Hepatitis B core antigen (HBcAg) is composed of a phosphorylated capsid, or C, protein with an icosahedral structure with subunits clustered as a dimer. The HBV genome is a circular, partially double-stranded, 3.2-kilobase DNA molecule having a highly compact coding structure, with every nucleotide in a coding region and more than half of the genome translated in more than one open reading frame (ORF) [18]. The S-ORF, including pre-S and S regions, codes for the viral surface proteins. The ORF precore codes for a hydrophobic peptide that directs the transplantation product to the endoplasmic reticulum, where further protein cleavage forms the third HBV antigen, HBeAg. HbeAg is a highly reliable clinical serum marker for the presence of infectious virion [19]. On a more basic level, HBeAg may serve as an immunoregulatory protein [20,21]. The
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P gene codes for the viral polymerase (pol), which overlaps the entire length of the S-ORF. The pol has three domains that catalyze genome synthesis (reverse transcriptase domain) [15]; degrade pregenomic RNA, facilitating replication (Rnase H domain) [16]; and affect encapsidation and initiate negative strand synthesis (terminal protein domain) [17]. The fourth ORF codes for HBX, a protein whose exact function is not known. HBX may have multiple effector pathways and may also counteract the increased proteolytic function of the infected cells, ensuring the reliability of the replication process. Hepadnaviral infection begins with the attachment of mature virions to host cell membranes, involving the pre-S domain and other factors. Entry of the virus results from fusion of the viral and host membranes and release of the complete virus, the nucleocapsid, into the cytoplasm. DNA replication occurs through an RNA intermediate using viral-encoded reverse transcriptase [14,22]. Although there is only one major serotype of HBV, HBsAg has five major subtype determinants: a, d, y, w, and r. All HBsAg-positive sera contain determinant a; d and y are mutually exclusive, as are w and r. Therefore, four subtype patterns are seen: adw, ayw, adr, and ayr, although ayr is quite rare. The subtypes have beenassociated with the geographic distributions of HBV, but do not seem to correlate with clinical course. Subtype adw is most common in the Western Hemisphere and Europe, and adr prevails in most of the Far East. HBV is not cytopathic for hepatocytes, and viral hepatitis is caused by the cellular immune response to HBV-infected liver cells [23 – 25]. Cytokines emanating principally from cytotoxic T lymphocytes, but also from helper T cells, natural killer cells, and natural killer T cells, inhibit HBV expression by activating liver cells to degrade viral RNA while also inhibiting HBV replication by preventing the assembly of or disrupting the nucleocapsid particles within which replication occurs. Cytotoxic T cells kill hepatocytes via Fas ligand and perforin-induced apoptosis. Epidemiology of HBV HBV is transmitted primarily from infected serum or blood products. HBsAg and HBV-DNA can be detected in a variety of body fluids and secretions, the importance of which is unknown. In homosexual men, transmission is most often by oral or genital contact or from asymptomatic bleeding lesions in the rectal mucosa. Transmission to the fetus occurs during pregnancy.
Hepatitis D HDV is the smallest known genome to infect humans [26], encoding just one protein, the hepatitis delta antigen (HDAg) [27,28]. HDV depends on HBV as a helper. The HBV envelope is composed of lipid and HbsAg, with a ribonucleoprotein core consisting of HBV genome RNA complexed with HDAg. At least three phylogenetically distinct genotypes exist, varying in geographic distribution and clinical manifestations [21,29 – 31]. Genotype 1, the most prevalent, found in
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North America, Europe, the South Pacific, Asia, and Africa, is associated with a broad spectrum of chronic disease. Genotype 2, found only in East Asia, is associated with a mild form of HDV disease. Genotype 3, found in northern South America, causes a severe hepatitis. Acute coinfection, generally a self-limiting disease, occurs in patients not previously infected with HBV. Almost all patients recover completely, but some progress to chronic HBV-HDV infection, with eventual cirrhosis. A few of these patients progress to chronic hepatitis with persistence of both viruses. HDV superinfection occurs in individuals previously infected with HBV. The course of infection is more rapid than in coinfection, always with severe hepatitis. In as many as 20% of cases, HDV superinfection progresses to chronic HDV. In approximately 70% of cases, there is chronic HBV-HDV and ultimate cirrhosis [13]. HDV, in liver transplant recipients with HBV and HCV coinfection, is associated with suppression of HCV replication and only mild inflammatory activity [32]. With better control of HBV, HDV infection is declining in incidence [28,33] Hepatitis C Pathogenesis and molecular biology HCV is a member of the Flaviviridae family of viruses. Falviviridae are small, enveloped viruses containing a positive-sense, single-stranded RNA genome. This family includes the viruses of yellow fever, dengue, bovine viral diarrhea, and swine fever. HCV is the only member of the genus Hepacivirus. HCV is genetically heterogeneous, with six major genotypes worldwide and more than 100 subtypes. The genomes of the most different HCV isolates differ by up to 35% [34 – 37]. In the United States, almost half of the isolates are genotypes 1a and 1b. Considerable genotype variation is found among different population groups. The HCV genotype also is a factor in response to therapies. For example, patients with genotype 1b, particularly prevalent in the United States, respond poorly to both interferon and combined interferon-ribavirin when compared with patients infected with genotype 2 or 3. HCV circulates as quasispecies that result from mutations accumulated over time and present at the time of infection. Such mutations probably enable HCV to replicate efficiently or resist immune mechanisms. Quasispecies complexities influence the outcome of acute hepatitis or the severity of persistent hepatitis. The response to therapy is also affected [37]. The genetic complexity has hampered vaccine development. HCV viral genomic RNA is associated with the capsid protein (C) to form the spherical, 30 nm nucleocapsid, which is surrounded by a lipid-containing envelope with two viral glycoproteins (E1, E2), derived from host membranes, to form the infectious virion. E1 and E2 proteins exhibit a high degree of genetic heterogeneity. The highly variable N-terminus of the E2 protein has been designated the hypervariable region 1 (HVR-1). HVR-2 is found within the
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envelope E2 protein of genotype 1b. HVR-1 may represent a neutralization epitope for humoral immunity. Indeed, the presentation of antibodies against HVR-1 can be followed by the emergence of new variants, against which antibodies may not be identifiable. The core protein of HCV is highly conserved with several B-cell epitopes that contributed to the development of practical serologic tests for HCV. Core protein C-terminus has a hydrophobic region that aids transport to the endoplasmic reticulum and membrane-dependent core processing. The translocation of core into the nucleus has been suggested as a mechanism of cell transformation [16]. The mechanism by which HCV enters and infects cells is not known. Most likely, E1 and E2 are involved in receptor binding with subsequent fusion with the host cell. HCV may form lipoprotein complexes and enter cells by endocytosis via lipoprotein receptors [16]. The specific role of lymphocytes remains to be elucidated [38]. Epidemiology of hepatitis C HCV is transmitted in a variety of ways, including transfusion of blood products [39,40]. HCV is food-borne and endemic in most countries [41]. Transfusion-related transmission is still common throughout the world, although declining rapidly in the United States. Transmission also occurs with unsafe injections and can also occur with hemodialysis. Occupational exposure is uncommon, and the prevalence of HCV infection among health care workers is no greater than that found in the general population. The role of sexual activity remains controversial. In a small group of people, a specific route of infection cannot be identified.
Liver biopsy findings Acute viral hepatitis The histopathologic features of the viral hepatitides are similar, regardless of cause. In acute hepatitis with focal necrosis, morphologic changes are distinctive, although not pathognomonic. Portal tracts usually show varying chronic inflammatory cell infiltrate, but lobular changes are dominant. At low magnification, the biopsy of the liver in a patient with acute hepatitis has a disordered or ‘‘dirty’’ appearance (Fig. 1). In fully developed acute hepatitis, changes are predominantly seen in zone 3 of the acinus (centrolobular and perivenular), and to a lesser extent in zone 1 (portal and periportal). Zone 3 hepatocytes show ballooning and degenerative change, with apoptotic cell necrosis and acidophilic (Councilman-like) body formation (Fig. 2). Ballooned hepatocytes have distended, pale, eosinophilic, and finely granular cytoplasm. Nuclei are hyperchromatic and vary in size. Acidophilic bodies result from apoptosis genetically programmed cell death, at least partly controlled by
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Fig. 1. Acute hepatitis B. The liver has a disordered appearance, partially obscuring the usual architecture, with irregularly distributed lymphocytes, swollen hepatocytes, and scattered acidophilic bodies.
the bcl-2 oncogene. Acidophilic bodies can be numerous, and are recognized as refractile, deeply eosinophilic bodies, sometimes with fragments of the nuclear chromatin of the dying hepatocyte. They are commonly surrounded by a mostly T-lymphocyte infiltrate, implicating a role for immunologically mediated cell injury and death [42,43]. Acidophilic bodies can also be seen without lymphocytes (‘‘naked acidophilic bodies’’ or ‘‘tombstones’’). Histiocytes and occasional plasma cells are also seen [43,44]. In contrast to hepatitis B and C, acute hepatitis A may be rich in plasma cells, sometimes mimicking AIH [15]. Reactive Kupffer cells phagocytose debris and are also prominent, whatever the cause.
Fig. 2. Acute hepatitis B, showing two acidophilic bodies (arrows).
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After cell death and removal of cell products, areas of ‘‘drop-out’’ are seen in which the liver plate structure is temporarily maintained despite the loss of one or more hepatocytes; this is easily shown with reticulin stain. Kupffer cells contain periodic acid-Schiff (PAS)-positive material, best appreciated after diastase digestion (dPAS) to remove liver cell glycogen. PAS-positive Kupffer cells and small aggregates of PAS-positive macrophages in portal tracts, although entirely nonspecific, can be indicators of recent hepatitis when cell necrosis and inflammation are not obvious. The loss of many liver cells causes extensive architectural disarray of liver cell plates, with a range of histologic patterns, including confluent, bridging, or submassive and massive necrosis; these changes are also well highlighted with reticulin stain. Acute viral hepatitis with confluent necrosis is the term used when focal necroses become numerous, coalesce, and cause cell loss in large areas of liver parenchyma. When confluent necrosis involves multiple acini, it takes the form of acute hepatitis with multiacinar (panacinar, submassive, massive) necrosis. Bridging necrosis often involves all three acinar zones, forming necroinflammatory connections between portal and perivenular areas (portal-central or portalto-portal bridging necrosis; Fig. 3). The pathogenetic mechanisms of these two types of bridging necrosis may be different. Furthermore, perhaps because of vascular shunting, portocentral bridging necrosis has a poorer prognosis in both acute and chronic hepatitis. In acute hepatitis, this pattern is associated with a high risk for progression into chronic hepatitis and ultimately cirrhosis. Some patients, particularly those with extensive bridging necrosis, die within weeks to months after the onset of hepatitis with subfulminant liver failure. Complete recovery, however, can also occur. When there is necrosis of many adjacent liver cells, the underlying reticulin network undergoes ‘‘collapse’’ (Fig. 4). Collapse, when shown with hematoxylineosin –stained tissue, is often indistinguishable from newly formed fibrous septa.
Fig. 3. Acute hepatitis B, with bridging necrosis and inflammation.
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Fig. 4. Acute hepatitis B, with an area of collapse (reticulin).
Reticulin stain highlights the compressed network of closely arranged reticulin fibers. True cirrhotic septa include elastic fibers, demonstrated with Victoria blue or orcein methods, as well as with other stains. In recent collapse, there are no elastic fibers [45]. Multinuclear, giant cells, formed by the fusion of hepatocytes, are shown in Fig. 5. They occur in children (giant-cell hepatitis), and are associated with a variety of infectious and noninfectious etiologies; giant cells are also seen in acute viral hepatitis in adults, including HCV [46]. Portal tracts are generally mildly expanded, with an infiltrate of lymphocytes, histiocytes, some plasma cells, and, sometimes, a few polymorphonuclear
Fig. 5. Acute hepatitis C, showing multinucleation of hepatocytes.
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leukocytes (PMNs) and eosinophils. If PMNs and/or eosinophils predominate, causes other than viral should be considered, especially drugs or toxins. Interface hepatitis (piecemeal necrosis) is characterized by inflammatory cells spilling into the limiting plate hepatocytes. Interface hepatitis is associated with a greater potential for progression and ultimate development of chronic hepatitis but is not as important as confluent necrosis. Viral hepatitis severity and progression is related more to the cause of the hepatitis than the inflammation pattern. Acute hepatitis A and E can have severe liver inflammation, including prominent interface hepatitis, but neither progresses to chronic liver disease. Hepatitis C often has a relatively mild inflammatory cell component, but has a high propensity for chronicity [14]. Cholestasis also may be seen, but it is generally mild and confined to canaliculi. True cholestatic variants of hepatitis can occur and may be prolonged.
Chronic hepatitis Chronic hepatitis B Chronic hepatitis B shows a full spectrum of histologic changes, including those described as ‘‘chronic persistent’’ and ‘‘chronic active hepatitis,’’ terms no longer useful, with progression to cirrhosis [47]. With active virus replication, portal and/or lobular hepatitis of varying degree is always present [48]. HBcAg can be immunohistochemically demonstrated in hepatocyte nuclei (Fig. 6) [49]. The 25-nm core particles of HBcAg correspond to the inner Dane particle compartment [50]. With increasing inflammation, core antigen may become obvious in the cytoplasm [51]. The e antigen, characteristic of acute hepatitis, can
Fig. 6. Recurrent hepatitis B, after orthotopic liver transplantation, showing abundant HBcAg (immunoperoxidase). (See also Color Plate 1.)
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also be seen. The e antigen in the serum is associated with HBV replication, correlating with serum HBV-DNA and DNA polymerase, and hepatocytes HBcAg. The e antigen expresses in both nucleus and cytoplasm. HbsAg can also accumulate in the replicative stage, demonstrable histochemically, with Victoria blue or orcein (Shikata), or immunohistochemically. Portal hepatitis (‘‘chronic persistent’’) is generally seen during the phase of low virus replication. Portal or lobular hepatitis may reoccur with either virus reactivation or superinfection with another virus, such as HDV [51]. In HBsAg carriers, in the absence of active replication, the liver biopsy may lack inflammation or there may be mild portal hepatitis. Typically, ground-glass hepatocytes containing HBsAg may be seen (Fig. 7). Ground-glass cells are slightly enlarged with increased, finely granular, pale, and eosinophilic cytoplasm. With formalin fixation, a clear halo separates the granular cytoplasm from the nuclear membrane. Ground-glass cells may be widely dispersed, may occur in nodular clusters, or may be so sparse as to be almost unrecognizable with both hematoxylin-eosin and Victoria blue (Fig. 7) or orcein, and identified only with immunohistochemical methods. When cirrhosis develops, the inflammatory component may disappear, although ground-glass cells may persist. Chronic hepatitis D When HBV and HDV infection occur at the same time, the patient may have an acute hepatitis, which is sometimes fulminant. Replication of HDV may occur without HBV replication, however, and HDV infection may occur in HBsAg carriers who do not have active HBV infection. Chronic HDV hepatitis occurs in this setting [13]. However, HBV must be present before HDV causes recognizable cell injury and disease [27].
Fig. 7. Chronic hepatitis B, carrier state, showing (left) ground-glass hepatocytes and (right)HBsAg with Victoria blue stain. (See also Color Plate 2.)
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There are no specific clinical features that identify patients with chronic HDV [52]. The diagnosis should be considered in any HbsAg-positive patients, but especially in cases of HBV chronic hepatitis that rapidly progress to cirrhosis, in known chronic HBV patients whose clinical course suddenly worsens, and in patients from areas (Brazil, Venezuela, southern Italy, the Middle East) where HDV is prevalent. HDV occurs only sporadically in North America and affects mostly high-risk groups, such as hemodialysis patients, intravenous drug users, hemophiliacs, homosexuals, and prison inmates. HDV may also rarely be transmitted in blood or blood products used for transfusion, when screening tests fail to detect HBsAg. When infection with HDV is accompanied by HBV, there is always significant histologic injury [53 – 55]. Sometimes only portal hepatitis is seen. Generally, the biopsy shows more severe inflammation and severe liver disease, progressing to either liver failure or cirrhosis. There are no specific histologic changes that differentiate HDV from HBV. It has been suggested that a fatnegative, foamy, cytoplasmic degeneration is indicative of HDV [54], but this has not proven to be specific. Chronic hepatitis C The cloning and sequencing of HCV, the agent responsible for most of the cases of non-A, non-B hepatitis, as well as many cases of ‘‘cryptogenic’’ cirrhosis, and the subsequent development of a clinically useful assay [4,56] were major triumphs of the new discipline of molecular biology. Using polymerase chain reaction, HCV is recoverable from virtually all cases of chronic HCV, including cirrhosis, but not from AIH or cryptogenic cirrhosis [57,58] Chronic HCV infection occurs all over the world, and is more common than HBV in North America [9,11]. As many as 40% to 50% of patients will progress to chronic hepatitis, and as many as 20% of these individuals will become cirrhotic [7,16,59 – 61]. A series of articles has helped define the key histologic features of chronic HCV [50,59,62 –74]. The key features of chronic HCV are as follows: (1) marked and patchy expansion of portal tracts by predominantly lymphocytic infiltrate with minimal spilling over (piecemeal necrosis) into adjacent lobules, often with a well-defined lymphoid aggregate, including true follicle formation with a welldefined germinal center; (2) varying degrees of bile duct damage and focal bile duct loss [66]; (3) varying degrees of steatosis, including micro- and macrovesicular fat; and (4) sinusoidal cell hyperplasia. Steatosis may be associated with accelerated disease [16]. The low-magnification appearance of the liver biopsy is characteristic: patchy enlargement of portal tracts, often in a distinctly globular form (Fig. 8), with partially or well-developed lymphoid follicles, minimal piecemeal necrosis, prominent sinusoidal cells, and little to moderate steatosis. Less commonly seen features include (1) slight lobular necrosis, (2) liver cell dysplasia, (3) multinucleation, and (4) accumulations of Mallory-like material in
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Fig. 8. Chronic hepatitis C, showing globular expansion of a portal tract with minimal interface hepatitis and mild lobular hepatitis. There is also minimal macrovesicular steatosis.
hepatocytes. Liver cell regeneration is generally not appreciated in routine sections in early stages, but can often be demonstrated immunohistochemically. The previously described features are not pathognomonic and have, individually and in groups, been recognized in other forms of chronic hepatitis, including HBV; however, they are most often seen in HCV. The constellation of the first set of features described, not uncommonly seen as a group, is virtually diagnostic. Differential diagnosis includes other forms of chronic hepatitis, including AIH. Generally, true lymphoid follicles are not seen in those conditions, and other features, such as piecemeal necrosis, severe lobular necrosis and inflammation, and parenchymal collapse, are prominent. Immunohistochemical and molecular methods, including in situ polymerase chain reaction (PCR), for identifying HCV in tissue are not yet widely available, although they have been evaluated in the research setting. The ‘‘gold standard’’ for diagnosis has been the identification of viral RNA in tissue homogenates using PCR [57,75]. Laser-capture microdissection with subsequent PCR has shown HCV to concentrate in zone 1 [76]. The chronic hepatitis report: grading and staging The terms for chronic hepatitis developed in the 1970s and 1980s to express prognostic judgments about liver biopsy findings (chronic active hepatitis [CAH], chronic persistent hepatitis [CPH], chronic lobular hepatitis [CLH]) are no longer useful or acceptable, particularly because they do not reliably represent the biologic behavior of the disease [77]. For example, the high virus replicative stage of beginning chronic HBV, when HBeAg and high levels of HBV-DNA are demonstrable in serum, may show great variation in histologic appearance; there may be changes of mild CAH, CLH, or even CPH [78 –81]. This is followed by the stage of seroconversion, with anti-HBe appearing in the serum; severe CAH
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with considerable lobular activity is generally seen, although there may be either CAH or CPH with either virus reactivation or superinfection with another virus, such as HDV [53]. Finally, if cirrhosis does develop, the inflammatory component may disappear entirely. Chronic hepatitis biopsies should be reported in a way to (1) convey the diagnosis of chronic hepatitis itself; (2) portray the degree of inflammation; (3) describe the extent, if any, of fibrosis; and (4) indicate, if possible, the cause. A variety of numerical schemes have been developed over the years. The best known of these is the Knodell approach [82] that, like the others, suffers from a lack of reproducibility and a lack of widespread acceptability [77,79,83]. If a scoring system is used, instead of simply using English-language terms, the report should identify the system and provide the scale of scoring. English language, without numerical scoring, remains acceptable and is still widely used by hepatopathologists [77]. The reproducibility of numerical systems increases, of course, with the numbers of cases studied and can be best used in large centers with many cases. The principal purpose of biopsy grading and staging is to document the specific state of potentially progressive disorders, such as the chronic hepatitides, and also to help hepatologists determine whether antiviral therapies should be applied. A mutually understandable reporting format is the key to achieving these goals. Recurrence after liver transplantation Hepatitis B and C will recur in the transplanted liver, if appropriate preventive therapy is not applied. The histologic features of recurrent disease are similar to those of naturally occurring disease, although in the early stages of recurrent hepatitis C, differentiation from early acute allograft rejection may be problematic [70]. The topic of liver transplantation is discussed in another article in this issue.
Hepatocarcinogenesis Hepatocellular carcinoma (HCC) accounts for as many as 500,000 deaths per year worldwide [84] and is discussed in detail elsewhere in this issue. However, there is widespread agreement that HBV plays a major role in the development of HCC, with the risk of developing HCC 20 times greater in HBsAg-positive individuals than in seronegative controls [85 –87]. Strong epidemiologic evidence and animal models [88] confirm this. In addition, HBV-DNA has been found integrated in both tumor cells and adjacent liver tissue in the vast majority of HCC patients with demonstrable HBsAg, as well as in a few of those who are HBsAg-negative [89,90]. There is currently no conclusive evidence that HBV is directly oncogenic. Instead, it is thought that chronic HBV directly induces HCC by either activating cellular oncogenes or by inactivating tumor suppressor genes, or indirectly as a consequence of chronic liver injury, inflammation, and regeneration. Integration of HBV-DNA can also induce carcinogenesis.
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The risk of developing HCC is approximately 25 times greater for HCV carriers compared with noncarriers [86,91,92]. In hepatitis C, it has been shown that transgenic mice expressing the HCV core protein develop HCC [93], but the precise basis for HCC in chronic HCV infection is not known.
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