Viral Hepatitis

Roger K. Moreira, MD

Douglas A. Simonetto, MD

8.1 GRADING AND STAGING OVERVIEW

In a fashion somewhat analogous to tumor pathology—where grade refers to features that reflect disease aggressiveness and stage refers to how advanced the disease is—grading and staging of various forms of liver diseases are a widely adopted practice around the world. Other disease categories, such as fatty liver disease, which show biologically distinct forms of fibrosis progression, have their own specific grading and staging systems.

Regardless of the specific method that is used, grading and staging systems provide prognostic value and are important tools when evaluating the effectiveness of new therapeutic modalities for chronic hepatitis. In clinical practice, most pathologists provide a descriptive term for grading (no activity, mild, moderate, or severe activity) and for staging (no fibrosis, portal/periportal fibrosis, bridging fibrosis, or cirrhosis). In our practice, we generally use a four-tiered staging system (Batts-Ludwig) for biopsies of chronic viral hepatitis (Figs. 8.1, 8.2, 8.3, 8.4, and 8.5).

Adequacy criteria

Although most forms of chronic viral hepatitis represent diffuse, relatively homogeneous processes, in some cases there can be challenges with regard to grading and staging. First, subcapsular samples should be interpreted with caution when they are superficial. The subcapsular area commonly shows nonspecific fibrous septa extending from the liver capsule into the hepatic parenchyma. This is usually restricted to tissue a few millimeters deep to the capsule but can mimic true advanced fibrosis with biopsies that are both superficial and tangential to the capsule. The subcapsular area may also show nonspecific inflammation that does not reflect the status of the remainder of the liver.

When needle core biopsies—the preferred type of sample for grading and staging—are performed, specimen adequacy evaluation should focus on two parameters: fragmentation and number of portal tracts. Even though no universally accepted consensus exists regarding the specific characteristics of an adequate sample, the overall literature seems conclusive about larger samples being better for grading and staging.¹ One study² suggested a minimum of 11 complete portal tracts, preferably

in a specimen containing two linear centimeters of tissue and cores being at least 1.4 mm (corresponding to a 16G needle) in thickness, is optimal to avoid underestimating both grading and staging. Fine needles (21G or less), although appropriate for the evaluation of focal lesions (i.e., tumors), have been found to systematically underscore grading and staging histologic variables.³ Ultimately, these data represent general and useful guidelines, but the final assessment of adequacy is rendered on a case-by-case basis, because even very small samples can sometimes provide all the information that is clinically needed for management purposes. A summary of potential pitfalls in grading/staging liver biopsy samples is presented in Table 8.1.

Figure 8.1 Stage 0 of 4. Small, normal portal tracts with no fibrosis.

Figure 8.2 Stage 1 of 4. Mild fibrous expansion of portal tracts.

Figure 8.3 Stage 2 of 4. Enlarged, irregular portal tracts with focal periportal septa.

Figure 8.4 Stage 3 of 4. Bridging fibrosis without parenchymal nodularity.

Figure 8.5 Stage 4 of 4. Extensive bridging fibrosis with parenchymal nodularity, i.e., cirrhosis.

The first scoring system to be devised was the “histologic activity index” (HAI), by Knodell and colleagues in 1981.⁴ This pioneering system, however, incorporated features that are now known to represent both grading and staging parameters into a single score. Nonetheless, the individual histologic features, namely, portal inflammation, “piecemeal” necrosis (interface hepatitis), focal (spotty) lobular necrosis, and confluent necrosis, along with staging parameters, have formed the basis for all major systems that were subsequently developed: Ishak [modified HAI] system, Scheuer system, Batts-Ludwig system, and the METAVIR system (see Tables 8.2, 8.3, 8.4, and 8.5).

Table 8.1 Potential pitfalls in grading and staging liver biopsy samples

Cause	Pitfall
Disease heterogeneity	Biliary tract disease (especially PSC and CF) and venous outflow impairment (especially congestive heart failure) can show heterogeneous fibrosis/cirrhosis, leading to both under- and over-staging
Sample size	Under-grading and under-staging in small samples
Biopsy location	Presence of nonspecific subcapsular fibrous septa and inflammation leading to overestimation of grade and stage
Bridging necrosis	Cases of severe parenchymal injury with bridging necrosis are often difficult to reliably differentiate from advanced fibrosis or cirrhosis
Macronodular cirrhosis	Fibrous septa can be very inconspicuous or absent in these cases; subtle parenchymal nodularity with surrounding thin fibrous rims should be carefully searched
PSC, primary sclerosing cholangitis; CF, cystic fibrosis.

All current staging systems are generally comparable, with relatively minor individual strengths and weaknesses. All have acceptable reproducibility. The choice among the different systems will largely depend on local preferences and purposes—with systems using fewer fibrosis stages being more reproducible and hence generally utilized for routine clinical diagnosis. Systems based on higher numbers of fibrosis stages provide more detailed information and are commonly used in research studies and clinical trials. Our group generally uses the Batts and Ludwig system in our daily practice.

Table 8.2 Scheuer system (1991)

Grade	Portal/periportal inflammation	Lobular inflammation
0	None or minimal	None
1	Portal inflammation	Inflammation without necrosis
2	Mild interface activity	Focal necrosis or acidophilic bodies
3	Moderate interface activity	Severe focal cell damage
4	Severe interface activity	Bridging necrosis
Stage	Degree of fibrosis
0	None
1	Enlarged, fibrotic portal tracts
2	Periportal or portal-portal septa but intact architecture
3	Fibrosis with architectural distortion but no obvious cirrhosis
4	Probable or definite cirrhosis
From Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol. 1991;13(3):372-374, with permission from Elsevier.

Table 8.3 Batts-Ludwig system (1995)

Grade	Lymphocytic piecemeal necrosis (interface activity)	Lobular inflammation and necrosis
0	None	None
1	Minimal, patchy	Minimal: occasional spotty necrosis
2	Mild, involving some or all portal tracts	Mild: little hepatocellular damage
3	Moderate involving all portal tracts	Moderate: noticeable hepatocellular damage
4	Severe, may have bridging necrosis	Severe: prominent diffuse hepatocellular damage
Stage	Degree of fibrosis
0	Normal connective tissue (no fibrosis)
1	Fibrous portal expansion (portal fibrosis)
2	Periportal fibrosis or rare portal-portal septa
3	Fibrous septa with architectural distortion; no obvious cirrhosis
4	Cirrhosis
From Batts KP, Ludwig J. Chronic hepatitis: an update on terminology and reporting. Am J Surg Pathol. 1995;19(12):1409-1417. http://journals.lww.com/ajsp/pages/default.aspx

Table 8.4 METAVIR system (1996) grading

Piecemeal necrosis, PN (0-3)	Lobular necrosis, LN (0-2)	Activity score, A (0-3)
0	0	0
0	1	1
0	2	2
1	0-1	1
1	2	2
2	0-1	2
2	2	3
3	0-3	3
The overall activity score (A) is reported as: no activity (A0), mild activity (A1), moderate activity (A2), and severe activity (A3).Staging
Stage	Degree of fibrosis
F0	No fibrosis
F1	Portal fibrosis without septa
F2	Portal fibrosis with rare septa
F3	Numerous septa without cirrhosis
F4	Cirrhosis
From Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996;24(2):289-293. doi:10.1002/hep.510240201

Table 8.5 Ishak system grading

A. Periportal or periseptal interface hepatitis (piecemeal necrosis)	Score
Absent	0
Mild (focal, few portal tracts)	1
Mild/moderate (focal, most portal tracts)	2
Moderate (continuous around <50% of tracts or septa)	3
Severe (continuous around >50% of tracts or septa)	4
B. Confluent necrosis
Absent	0
Focal confluent necrosis	1
Zone 3 necrosis in some areas	2
Zone 3 necrosis in most areas	3
Zone 3 necrosis plus occasional portal-central bridging necrosis	4
Zone 3 necrosis plus multiple portal-central bridging necrosis	5
Panacinar or multiacinar necrosis	6
C. Focal (spotty) lytic necrosis, apoptosis, and focal inflammation
Absent	0
One focus or less per ×10 objective	1
Two to four foci per ×10 objective	2
Five to ten foci per ×10 objective	3
More than ten foci per ×10 objective	4
D. Portal inflammation
Absent	0
Mild, some or all portal tracts	1
Moderate, some or all portal tracts	2
Moderate/marked, all portal tracts	3
Marked, all portal tracts	4
Staging
Stage	Degree of fibrosis
0	No fibrosis
1	Fibrous expansion of some portal tracts, with or without short septa
2	Fibrous expansion of most portal tracts, with or without short septa
3	Fibrous expansion of most portal tracts with occasional portal-portal bridging
4	Fibrous expansion of portal tracts with marked portal-portal as well as portal-central bridging
5	Marked bridging with occasional nodules (incomplete cirrhosis)
6	Cirrhosis, probable or definite
The overall grade is reported as a sum of individual scores (total score range: 0-18). From Callea F, De Grootes J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol. 1995;22(6):696-699, with permission from Elsevier.

8.2 HEPATITIS A

Etiology and pathogenesis

Hepatitis A is caused by a small, nonenveloped RNA virus belonging to the Picornaviridae family, which is spread almost exclusively by fecal-oral transmission, either directly or through contaminated food or water. Upon entry into the hepatocyte, the viral RNA is uncoated, viral proteins are synthetized, and the assembled viral particles are shed into the biliary system and excreted in the feces. Viral infection of hepatocytes is thought to have little or no direct cytotoxic effect, with liver injury stemming predominantly from immune-mediated attack.

Clinical manifestations

Hepatitis A virus (HAV) infection has three main clinical phases: incubation, symptomatic, and convalescence. The incubation period of acute hepatitis A varies from 14 to 50 days. The clinical manifestations also vary and are age dependent. Only 30% of children (age 6 or less) will develop symptoms, whereas more than 70% of adults develop jaundice with markedly elevated transaminases. Additional symptoms include fatigue, anorexia, nausea, vomiting, diarrhea, fever, headaches, and arthralgia.⁵ The symptomatic phase typically lasts 1 to 8 weeks and coincides with the presence of IgM anti-HAV antibodies. Positive IgM antibodies are used to confirm the diagnosis of HAV
infection and may persist for up to 6 months. Total anti-HAV IgG antibody levels rise in parallel with a decrease in IgM titers and reflect long-term immunity against hepatitis A.⁶ A small subset of patients with hepatitis A will develop severe forms of infection, including cholestatic hepatitis, relapsing hepatitis, and rarely, fulminant hepatitis.⁷,⁸

Histologic features and differential diagnosis

The histopathology of hepatitis A will largely depend on the severity of the clinical presentation. A large proportion of cases is either asymptomatic or associated with very mild signs/symptoms and hence is not biopsied. Sampled cases most commonly show prototypical features of an acute, lobular-predominant hepatitis (Fig. 8.6), with variable inflammation (mostly CD8+ T cells), numerous acidophilic bodies, lobular disarray, hepatocyte swelling, Kupffer cell hyperplasia, sometimes with hepatocanalicular cholestasis. Acute HAV infection can also show relatively dense portal inflammation with interface activity, sometimes with increased plasma cells,⁹ mimicking autoimmune hepatitis. This pattern can also be seen in cases with a clinically protracted or relapsing course. In a small minority of cases, fulminant hepatitis occurs, and the histologic picture is that of a massive/submassive necrosis (Fig. 8.7), with prominent regenerative changes and cholestasis of the residual hepatocytes, sometimes with striking ductular reaction.¹⁰

Prognosis and treatment

Hepatitis A infection will spontaneously resolve in over 99% of cases. The treatment is mainly supportive, including adequate hydration and avoidance of hepatotoxic drugs. Liver transplantation may be required in as many as 30% of patients with fulminant hepatitis A.¹¹ Hepatitis A is a vaccine-preventable disease, and the fundamental management of hepatitis A remains active immunization.

Figure 8.6 Hepatitis A. Acute hepatitis pattern, featuring a predominantly lobular process, with mild necroinflammation, disarray, and acidophilic bodies.

Figure 8.7 Hepatitis A. A case of fulminant hepatitis with massive parenchymal necrosis.

8.3 HEPATITIS B AND DELTA VIRUS

Etiology and pathogenesis

Although hepatitis B has a relatively low prevalence in the United States, an estimated 250 million people (approximately 3.6% of the world’s population) are HBsAg positive worldwide, representing one of the main causes of chronic liver disease. The prevalence of hepatitis B virus (HBV) infection is highly variable, being highest in sub-Saharan Africa and certain countries in the Middle-East, Asia, and Western Pacific region.¹² HBV is an enveloped DNA virus, a member of the Hepadnaviridae family. Transmission occurs through blood and body fluid exchange, taking place predominantly during the perinatal period (vertical transmission) or during childhood in high-prevalence areas. In low-prevalence areas, such as the United States and Western Europe, infections are acquired mostly during young adulthood through sexual contact or injection drug abuse (horizontal transmission). The natural history of the disease is also deeply influenced by the timing of initial infection. The infection becomes chronic in approximately 90% of cases in perinatal transmission, whereas the opposite is seen in infections acquired in adulthood, where over 90% of infections have spontaneous resolution.

Patients with chronic infection, especially those acquired during the perinatal period or early childhood
years, as well as those with high levels of HBV replication (high HBeAg and HBV DNA levels), have the highest risk for developing hepatocellular carcinoma.¹³ Several HBV genotypes have been described (eight in total, A-H), showing distinct geographic and ethnic distributions around the world, as well as different risk levels for the development of cirrhosis and hepatocellular carcinoma.¹⁴, ¹⁵, ¹⁶, ¹⁷ Finally, HBV viral replication occurs through reverse transcription but without an effective proof reading mechanism, leading to a high rate of viral mutations. Precore and basal core mutations¹⁸,¹⁹ have been linked to diminished or loss of HBeAg production, and YMDD mutants are associated with drug resistance.²⁰

Following acute infection, the disease goes through several phases, with different serological, histologic, and immunophenotypic characteristics (Table 8.6). In the initial phase—immune tolerance—there is a low level of immunologic response to the virus, variable but generally mild necroinflammatory activity, and high levels of viral replication, with corresponding serology that is HBeAg positive and anti-HBe negative. In the immune clearance phase, there is heightened immune response to the virus, and the histologic activity tends to be correspondingly high. This is followed by a low-replicative phase in which both viral replication and histologic activity are low. This sequence of events, however, is complex and variable from case to case. Disease activity can also flare above baseline for several reasons, including emergence of viral mutants, coinfection with delta virus, and treatment-related factors.

The hepatitis D virus (HDV, also called delta virus) is known as a “defective virus,” because it requires hepatocytes being also infected with HBV in order to replicate. HDV can be transmitted either as a coinfection with HBV or as a superinfection in patients with chronic hepatitis B. In cases of coinfection, HDV confers an increased risk of fulminant hepatitis,²¹ while superinfection has been associated with increased inflammatory activity (mimicking hepatitis B flares or other superimposed acute injuries). Higher rates of chronicity, increased liver-related mortality, and higher risk of hepatocellular carcinoma have also been reported with hepatitis D.²²

Clinical manifestations

Acute hepatitis B symptoms are nonspecific and mainly constitutional, including low-grade fever, fatigue, nausea, myalgia, and arthralgia. Approximately 70% of patients will present with subclinical or anicteric hepatitis. Jaundice, present in only 30% of acute HBV infections, typically begins within 10 days after the onset of symptoms. Marked transaminase elevations
are often seen, with levels over 1,000 to 2,000 IU/L, followed by hyperbilirubinemia. These symptoms usually resolve spontaneously within 1 to 3 months.

Table 8.6 Clinical features of different phases of chronic hepatitis B

Phases of chronic hepatitis B	Serologic markers	Findings
Immune tolerant phase	HBeAg positive	High-serum HBV DNA levels Normal aminotransferases Mild or no hepatic necroinflammation Mild or no fibrosis progression
Immune active or immune clearance phase	HBeAg positive	Low-serum HBV DNA levels Elevated aminotransferases Moderate-severe liver necroinflammation Fibrosis progression
Inactive HBV carrier state	HBeAg negative HBeAb positive	Low-serum HBV DNA levels Normal aminotransferases No hepatic necroinflammation No fibrosis progression
Reactivation phase	HBeAg negative HBeAb positive	Fluctuating levels of serum HBV DNA Elevated aminotransferases Mild-moderate liver necroinflammation Fibrosis progression
HBsAg-negative phase or occult HBV infection	HBeAg negative HBeAb positive HBsAg negative HBsAb negative	Undetectable serum HBV DNA Detectable HBV DNA in the liver At risk for reactivation with immunosuppressive therapy
HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBeAb, hepatitis B e antibody; HBsAb, hepatitis B s antibody; HBsAg, hepatitis B s antigen.

The vast majority of patients with chronic hepatitis B are asymptomatic, but acute exacerbations can manifest as fatigue, anorexia, nausea, and jaundice. The transaminase levels are often normal or only mildly elevated in chronic hepatitis B. Extrahepatic manifestations of chronic hepatitis B occur in 10% to 20% of patients and have been attributed to circulating immune complexes containing HBsAg. Extrahepatic manifestations include polyarteritis nodosa, membranous and membranoproliferative glomerulonephritis, and papular acrodermatitis.²³

The diagnosis of hepatitis B is based on serologic markers and confirmed by the presence of HBsAg and/or HBV DNA in the serum (Table 8.7).²⁴ The presence of HBsAg for longer than 6 months in the blood defines a hepatitis B infection as chronic. HDV exposure can be detected by the presence of IgG anti-HDV antibodies. The diagnosis of active HDV infection is confirmed by detection of serum HDV RNA by polymerase chain reaction (PCR).

Histologic features

Acute hepatitis B

Acute hepatitis B is rarely an indication for liver biopsies, but biopsies do occur when acute hepatitis B is not clinically suspected. The histologic findings in acute hepatitis B are similar to other causes of acute hepatitis, including a lobular-predominant process characterized by various degrees of necroinflammation. Ground-glass inclusions are absent in acute infection, and HBV-related immunohistochemical markers (see discussion later) are usually negative.

Table 8.7 Serologic markers in hepatitis B

Serologic marker	Interpretation
Hepatitis B surface antigen (HBsAg)	Current hepatitis B infection
Hepatitis B surface antibody (HBsAb)	Resolved hepatitis B infection or immunization
Hepatitis B e antigen (HBeAg)	Indicates high level of viral replication and infectivity
Hepatitis B e antibody (HBeAb)	Indicates HBeAg seroconversion and usually marks the transition to inactive carrier state
IgM hepatitis B core antibody (IgM anti-HBc)	Indicates acute hepatitis B or reactivation. The only positive marker during the window period (between disappearance of HBsAg and appearance of HBsAb)
IgG hepatitis B core antibody (IgG anti-HBc)	Indicates past or current HBV infection