Testing for Viral Hepatitis A Practice Parameter

AJCP / TESTING FOR VIRAL HEPATITIS Testing for Viral Hepatitis A Practice Parameter Ronald A. Sacher, MD, FRCPC, 1 Stephen M. Peters, PhD, FAAM, 2 and John A. Bryan, MD 3 Key Words: Viral hepatitis; Laboratory testing; Guidelines Hepatitis is defined broadly as liver parenchymal inflammation. The differential diagnosis of this inflammation is extensive and includes a wide variety of infections, drug and toxin exposures, vascular disease, and metabolic and immune derangements. This practice parameter focuses on viral hepatitis Table 1 ; however, other causes of hepatitis must be excluded in the workup of a patient with evidence of inflammatory liver disease. Indeed, in view of the prevalence of viral hepatic infection, the presence of serologic markers of hepatitis may not represent the only cause of the patient s illness. Laboratory evaluation is a necessary adjunct in the assessment of the patient with liver disease and complements the history and physical examination. In the evaluation of hepatic disease, laboratory test results can indicate parenchymal injury; biliary injury; abnormalities of bilirubin metabolism, excretory function, or synthetic function; evidence of immunologic damage; hepatic neoplasia; and viral hepatic diseases. These categories can be expanded to include several specific blood tests, such as measurement of alpha 1 -antitrypsin, antinuclear antibodies, anti smooth muscle antibodies in autoimmune hepatitis, antimitochondrial antibodies in primary biliary cirrhosis, iron studies in hemochromatosis, and ceruloplasmin in Wilson disease. insidious onset. Hepatitis C, also insidious, usually is asymptomatic and has a subclinical protracted course in most cases. The most characteristic laboratory findings in acute viral hepatitis are elevation of the serum transaminase levels, which may reach 100 times their normal ranges. 1 Direct and indirect bilirubin levels usually are elevated in roughly equal proportions in icteric patients. The prothrombin time most often is normal; if significantly elevated (>4 seconds beyond upper limit of reference range), it portends hepatic failure and a poor prognosis. Alkaline phosphatase and lactate dehydrogenase levels are normal or only mildly elevated. In practical terms, chronic hepatitis is defined as the presence of elevated liver transaminase levels for longer than 6 months, although levels may fluctuate between normal and elevated, especially in hepatitis C infection. The diagnosis of viral hepatitis depends on the identification of serologic markers, typically detected by enzyme immunoassays. 2 These assays are automated readily and, therefore, are relatively fast and inexpensive. The results are semiquantitative; the presence or absence of an antibody or antigen is reported without quantification of titers. In selected cases, viral genomic assays can be performed. Pathophysiology and Clinical Pathology The history and physical examination findings are, for the most part, nonspecific in patients with viral hepatitis but may suggest risk factors for hepatitis A (eg, travel to an endemic area, ingestion of contaminated food or shellfish), hepatitis B (eg, intravenous drug use, homosexuality, occupational exposure), or hepatitis C (eg, multiple transfusions or intravenous drug abuse). Clinically, hepatitis A and B are difficult to distinguish from one another. Hepatitis A, however, has an abrupt onset, whereas hepatitis B has an Hepatitis A Hepatitis A is a nonenveloped RNA virus that causes an acute self-limited illness characterized by jaundice, fever, Table 1 Differential Diagnosis of Chronic Hepatitis in Adults Hepatitis B, C, D Chemical injury, drugs, and alcohol Autoimmune hepatitis Hemochromatosis Alpha 1 -antitrypsin deficiency Wilson disease 12 Am J Clin Pathol 2000;113:12 17 American Society of Clinical Pathologists

AJCP / SPECIAL ARTICLE anorexia, and diarrhea. 3 The virus is transmitted almost exclusively through the fecal-oral route. Clinical severity is related directly to the age of the patient. Acute infection is confirmed by detection of IgM anti hepatitis A virus (HAV), which appears early in the course of infection while transaminase levels are still elevated and viral shedding is still occurring. Rheumatoid factor potentially can cause false-positive results. IgG anti-hav becomes the predominant antibody during convalescence and persists throughout life; measurement of IgG anti-hav serves little clinical purpose other than to confirm previous infection. Rather than directly assaying IgG anti-hav, the primary test available is total anti-hav, which measures antibodies of the IgG, IgA, and IgM classes. This test has been used as a screening test for HAV infection, although confirmation of acute infection still requires detection of IgM antibodies. The recent development of a vaccine for HAV will result in positive test results for total anti-hav (specifically IgG) in immunized persons. 3 Hepatitis B Hepatitis B is a DNA virus with a complex structure consisting of a nucleocapsid core containing predominantly double-stranded DNA and DNA polymerase covered with an outer protein coat, which contains the surface antigen (HBsAg). Hepatitis B virus (HBV) infection is prevalent throughout the world, especially in Asia, where prevalence may be as high as 20% of the total population. Infection leads to several possible clinical outcomes. The frequency of symptomatic acute hepatitis B increases with increasing age, while the risk for chronic infection decreases. 3,4 Thus, fewer than 5% of neonates infected with the virus have symptoms of acute hepatitis, while chronic infection develops in up to 90%. Overall, roughly two thirds of persons infected with HBV remain asymptomatic, symptomatic acute hepatitis develops in one fourth, and the remainder become lifelong carriers. Chronic carrier status is associated with an increased risk for the development of cirrhosis and hepatocellular carcinoma. 4 Active HBV infection usually is confirmed by the presence of HBsAg. HBsAg is the first serologic marker to appear, preceding elevation of transaminase levels and persisting throughout the icteric and symptomatic phase of acute infection. Typically, HBsAg becomes undetectable 1 to 3 months after the onset of jaundice and rarely persists beyond 6 months. After HBsAg disappears, antibody to HBsAg (anti-hbs) appears and persists indefinitely. Usually there is a hiatus between the disappearance of HBsAg and the emergence of anti-hbs. However, increased sensitivities of the antigen and antibody assays have reduced the incidence, length, and clinical relevance of this hiatus. Furthermore, antibody to the hepatitis B e antigen (anti-hbe) also is positive during this phase. If the HBsAg and anti-hbe are positive, viral replication has subsided, and resolution of the infection should occur shortly. Thus, the presence of anti- HBe also represents an immune status. 5 The temporal association between the appearance of anti-hbs and the resolution of symptoms, and the observation that the majority of persons with anti-hbs are protected from reinfection, led to the proposal that anti-hbs is the protective antibody against HBV infection. Anti-HBs rarely is present during acute infection, but low titers may occur during chronic infection. Current immunization protocols use HBsAg produced by recombinant DNA technology to induce anti-hbs antibodies. These antibodies are the only serologic markers present in the serum of immunized persons who have never been infected with the virus. In the vast majority of cases, this antibody persists for life; however, in patients with compromised immunity, booster immunizations, although controversial and not widely practiced, have been recommended when the anti-hbs titer falls below 10 miu/ml. Because hepatitis B core antigen (HBcAg) is sequestered within HBsAg, HBcAg is not routinely detectable in patients with HBV infection, but its derivative, the hepatitis B e antigen (HBeAg), can be measured. Antibody to the HBcAg (anti-hbc), on the other hand, is readily demonstrable, beginning 1 to 2 weeks after the appearance of HBsAg and preceding detectable levels of anti-hbs by weeks to months. Thus, anti-hbc is detectable during the period in which there is neither HBsAg nor anti-hbs and can serve as a marker for current or recent infection. In some patients, years after infection, anti-hbc remains detectable longer than anti-hbs, so that remote infection is manifest by the presence of anti-hbc in the absence of anti-hbs and HBsAg. The immunoglobulin class of anti-hbc can be used to distinguish between recent and remote infection. IgM anti- HBc predominates during the first 4 to 6 months after infection, whereas IgG anti-hbc predominates thereafter, in patients with complete recovery and in chronic carriers. IgM anti-hbc often persists in chronic infection, but in titers that are below the level of detection of most commercial assays. IgM anti-hbc can be used to establish acute infection in the rare patient (fewer than 5%) whose HBsAg is below detectable limits. Similarly, IgG anti-hbc could be compatible with defining infection in the rare carriers whose HBsAg is undetectable. IgG anti-hbc simply implies exposure to HBV, recent or remote in these cases. The other readily detectable HBV serologic marker is HBeAg, which appears concomitantly or shortly after HBsAg. Its appearance coincides with the phase of maximal American Society of Clinical Pathologists Am J Clin Pathol 2000;113:12 17 13

Sacher et al / TESTING FOR VIRAL HEPATITIS viral replication and reflects the presence of circulating intact virions, DNA polymerase, and HBV DNA. Thus, the principal relevance of HBeAg is as a marker of maximal infectivity. In addition, HBeAg is a marker of active infection. In acute self-limited infections, HBeAg disappears shortly after peak transaminase elevations and before the disappearance of HBsAg. At this point, anti-hbe becomes detectable. In protracted infections, HBeAg may persist, indicating continuing replicative infection. Because HBeAg is present invariably in active infections, testing for its presence is indicated primarily during follow-up of chronic infection. Conversely, the presence of anti-hbe and the absence of HBeAg in chronic HBV infection indicates that the infection probably is nonreplicative. Detection of HBV DNA in liver or serum or DNA polymerase in serum is available in a limited number of laboratories. As with HBeAg, HBV DNA and DNA polymerase serve as sensitive markers of HBV replication. The presence of HBV DNA is, in fact, the most sensitive marker for ongoing infection; quantification of the amount of DNA may have prognostic significance and has been used as an indicator of response to interferon-alfa-2b therapy. Hepatitis C Hepatitis C is a small, enveloped, single-stranded RNA virus. Most infections are asymptomatic; in the patients in whom acute hepatitis develops, the symptoms usually are milder than in acute infection with HAV or HBV. Unfortunately, chronic sequelae are relatively common. Chronic infection occurs in 50% to 80%, and cirrhosis develops in 20% to 30% of persons who have had chronic infection for more than 20 years. 6 Elucidation of the structure of the hepatitis C virus (HCV) has permitted production of recombinant antigens, to which most infected persons produce antibodies. These antibodies generally are nonneutralizing and nonprotective, but they serve as markers of infection. Several generations of immunoassays (enzyme-linked immunosorbent assays [ELISAs] and enzyme immunoassays) have been developed for the detection of these antibodies. First-generation assays were relatively nonspecific and nonsensitive. In addition, there often was an extended window period after initial infection during which the assay was negative (averaging 15 weeks). Indeed, some patients never developed antibody to the specific antigen incorporated in the test. Second-generation ELISAs, initially licensed in 1992, added 2 epitopes to the assay, with a resultant increased sensitivity. Antibodies to these epitopes arise earlier in infection, and the window period was shortened considerably, to less than 12 weeks. Third-generation tests have a slightly greater increase in sensitivity but have reduced the window period to only about 11 weeks. Suboptimal specificity is a problem with all of these assays. Most false-positive cases remain unexplained, although aged serum samples, hypergammaglobulinemia, rheumatoid factor, and recent immunization against influenza virus have been implicated as putative causes in specific cases. Because of this low specificity, ELISA-positive samples must be confirmed with recombinant immunoblot assay (RIBA). The RIBA incorporates critical epitopes of the virus on a nitrocellulose strip, which are then overlaid with patient s serum. Antibodies are detected with enzyme-linked antiglobulin antibody. A positive test result requires at least 2 bands; 1 positive band is interpreted as an indeterminate result. Incorporation of superoxide dismutase on the strip serves as an internal negative control. Two generations of RIBAs have been developed; a third, with several modified epitopes, recently has been licensed. It seems likely that with the high frequency of developing chronicity, HCV antibody would persist for a protracted time, and the viremia also would be lifelong because of the persistent infection. Reverse transcriptase polymerase chain reaction (RT- PCR) is the most sensitive but least practical method for detecting HCV and is available only in specialized laboratories. HCV RNA appears in the serum within days of exposure, before the increase in the alanine aminotransferase (ALT) level, and, thus, its detection by RT-PCR is the most effective method to determine infection. The majority of patients with chronic infection have abnormalities in ALT levels, which often fluctuate widely. About one third of patients with HCV with chronic infection have persistently normal serum ALT levels. There is inconsistent correlation between ALT levels and disease severity as judged histologically. Another method for detecting HCV RNA (or HBV DNA) in serum is branched DNA amplification. In this technique, serum HCV RNA is bound to synthetic oligonucleotides, which are attached to microtiter wells. Multiple copies of labeled branched DNA are then added, which bind to the HCV RNA; detection is with chemiluminescence. The branched DNA amplification assay is quite specific but is not as sensitive as RT-PCR. There also are some additional limitations of these molecular tests, since there are at least 6 HCV genotypes and considerable genetic heterogeneity. The conserved 5 untranslated region serves as the most frequent site for PCR detection of HCV-RNA. Molecular assays also can be used to determine viral load and response to antiviral therapy. 7 Hepatitis D Hepatitis D, also known as the delta agent, is a defective RNA virus that is absolutely dependent on previous or 14 Am J Clin Pathol 2000;113:12 17 American Society of Clinical Pathologists

AJCP / SPECIAL ARTICLE Hepatitis A Acute hepatitis B Acute hepatitis C R/O hepatitis A IgM anti-hav IgM Anti-HBc RIBA confirmation False + R/O hepatitis B HBsAg * R/O hepatitis C ELISA anti-hav Ab (third generation) EBV hepatitis R/O EBV IgM anti-ebv 4-Fold IgG EBV rising titer R/O other causes: drugs, alcohol, immune causes Figure 1 Simplified primary diagnostic algorithm for serologic testing in suspected acute viral hepatitis. (*Refer also to the supplementary algorithm, Figure 2.) EBV, Epstein- Barr virus; ELISA, enzyme-linked immunosorbent assay; HAV, hepatitis A virus; anti-hbc, antibody to hepatitis B core; HBsAg, hepatitis B surface antigen; R/O, rule out; RIBA, recombinant immunoblot assay. Acute exacerbation in chronic HBV infection HBeAg Acute HDV coinfection HBsAg *R/O hepatitis D (delta Hepatitis) anti-hdv + R/O EBV R/O CMV R/O HCV* Viral R/O other causes Nonviral Drugs Alcohol Immune causes Metabolic causes Figure 2 Supplementary diagnostic algorithm for serologic testing in suspected chronic viral hepatitis (rule out acute HAV, HBV, and HCV infection; see primary algorithm, Figure 1). *See text. CMV, cytomegalovirus; EBV, Epstein-Barr virus; HAV, hepatitis A virus; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HCV, hepatitis C virus; HDV, hepatitis D virus; R/O, rule out. concurrent hepatitis B infection. Coinfection usually leads to more severe acute disease and a higher risk of fulminant hepatitis than with hepatitis B alone. Superinfection often leads to deterioration in the clinical condition and markedly increases the risk of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Hepatitis D is rare in the United States. 4 The nucleocapsid core of the delta agent contains the RNA genome and expresses the delta antigen; this core is encapsulated by HBsAg. Delta antigen is present only transiently in serum, during the late incubation period. In addition, most enzyme immunoassays for this antigen have poor sensitivity; they almost always are negative. The presence of delta antigen can be confirmed by Northern blot analysis, which is available at a limited number of reference laboratories. Commercial assays are available for total (IgG and IgM) and IgM anti-delta antibody. IgM anti-delta predominates during acute infection; in self-limited illness, this antibody is low-titer and disappears before the clearance of HBsAg. Several samples may be needed to detect its presence. In contrast, IgM and IgG anti-delta circulate in high-titer in chronic delta infection. Hepatitis E Hepatitis E is an RNA virus found primarily in India, central and southeast Asia, and the Middle East. Rare cases have been reported in the United States in travelers from endemic areas. 8 In addition, recent outbreaks have been reported in Mexico. The pathogenesis is similar to that of hepatitis A, although clinical symptoms usually are more severe. Infection in pregnant women is especially dire, with a 20% to 30% mortality rate. Enzyme immunoassays exist for detecting IgM and IgG antibodies to different epitopes of the virus. These assays are not widely available, though, and suspected cases should be referred to the Centers for Disease Control and Prevention. Hepatitis G Hepatitis G (and the closely related hepatitis GB virus C) is a recently described member of the hepatitis viruses. It is transmitted parenterally and has been found in intravenous drug abusers, patients receiving dialysis, patients who have received transfusions, and transplant recipients. A high prevalence has been found in the blood donor population of the United States (roughly 1%-2%). The clinical significance of this virus is unknown; the vast majority of patients with acute hepatitis G have clinically silent disease 9 and have no evidence of liver disease. The peak ALT levels seen with chronic hepatitis G are half the values seen with hepatitis C. There are no serologic assays for hepatitis G; it is detected only in research laboratories by using molecular methods (RT-PCR for hepatitis G virus RNA). 9 Diagnosis Testing for viral hepatitis often involves ordering a panel of serologic tests, although selective test ordering based on index of suspicion is considered more appropriate by many. 1 A testing algorithm for acute hepatitis is outlined in Figure 1. A standard panel for acute hepatitis should include IgM anti-hav, HBsAg, IgM anti-hbc, and anti-hcv. 3 In American Society of Clinical Pathologists Am J Clin Pathol 2000;113:12 17 15

Sacher et al / TESTING FOR VIRAL HEPATITIS Table 2 Viral Hepatitis Serologic Findings Type Clinical Features Transaminases Acute Disease Immunity A (RNA) Acute self-limiting; fecal-oral Markedly elevated, 3+ to 4+ IgM anti-hav IgG anti-hav transmission; no carrier state B (DNA) Acute or chronic; often 1+ to 3+ (acute); IgM anti-hbc; HBsAg; IgG anti-hbs; anti-hbe asymptomatic; 5% infected variable; chronic HBeAg; HBV DNA blood and body fluids C (RNA) Usually chronic Mild to moderate elevation, Total anti-hcv; HCV RNA Unknown even in acute cases D (RNA) Requires HBV coinfection; Often markedly elevated IgM anti-hdv; total Unknown acute or chronic; rare in anti-hdv; may United States require multiple assays E (RNA) Epidemic similar to HAV; Variable Testing not widely Unknown acute self-limiting; water available; reference borne and enteric; laboratory: CDC transmission in India and Southeast Asia G (RNA) Close homology to HCV; high Variable HGV-RNA and EIA Unknown prevalence (2% of US donors); (reference laboratory usually mild disease, if at all; only) disease spectrum uncertain Non A-E, Possibly acute or chronic Variable None Unknown exclude EBV and CMV CDC, Centers for Disease Control and Prevention; CMV, cytomegalovirus; EBV, Epstein-Barr virus; EIA, enzyme immunoassay; HAV, hepatitis A virus; anti-hbc, antibody to hepatitis B core; anti-hbe, antibody to hepatitis B e antigen; HBeAg, hepatitis B e antigen; anti-hbs, antibody to hepatitis B surface antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HGV, hepatitis G virus. patients with fulminant hepatic failure or known infection with hepatitis B, anti hepatitis D virus (HDV) testing also should be ordered. The presence of antibodies to HAV or HCV may confirm acute or previous exposure. The absence of anti-hcv, however, does not rule out the possibility of HCV infection, as there is some delay after infection before the appearance of antibodies (sometimes even up to 1 year). IgM anti-hbc confirms acute hepatitis B, usually with the presence of HBsAg. If HBsAg is present, anti-hdv testing is appropriate in certain clinical settings, such as for patients with a recent history of travel to the Mediterranean, intravenous drug abuse, or multiple transfusions. HBsAg without IgM anti-hbc suggests chronic hepatitis B (either active or carrier status). For patients with suspected chronic hepatitis, tests should include HBsAg and anti-hcv. 4 If the HBsAg result is positive, further testing should include HBeAg, to determine whether active viral replication is present, and anti- HDV. At present, using second-generation anti-hcv tests, confirmatory testing should be done with recombinant immunoblot assay. A negative HCV test result should initiate a search for other causes of chronic liver disease, especially autoimmune hepatitis (Table 1). Concomitant infections with 2 or more of these viruses have been reported. Liver biopsy rarely is indicated in acute hepatitis. On the other hand, biopsy of the liver is necessary in chronic hepatitis to assess severity and to evaluate the necessity or justification for therapy. 3,4 Other viruses also can cause acute hepatitis. Cytomegalovirus and Epstein-Barr virus are the most common and lead to similar clinical disease. Serologic tests are helpful for distinguishing these infections. HIV, herpes simplex virus, varicella zoster virus, adenovirus, and coxsackie virus infections also can lead to hepatic inflammation. 3 Table 2 lists the clinical and laboratory comparison between the major viral types discussed in this parameter. A diagnostic algorithm for the evaluation of acute viral hepatitis is shown in Figure 1, and Figure 2 shows a supplemental testing algorithm for suspected chronic hepatitis. From the 1 Departments of Medicine, Pathology, and Laboratory Medicine; 2 Departments of Pediatrics, Pathology, Microbiology and Immunology and the Division of Infectious Diseases Department of Laboratory Medicine; Georgetown University Medical Center, Washington, DC; and 3 Department of Pathology, Elliot Hospital, Manchester, NH. Address reprint requests to Dr Sacher: Chairman, Laboratory Medicine Dept, Georgetown University Medical Center, 3800 Reservoir Rd, NW, Washington, DC 20007. This practice parameter represents the opinions and recommendations of the authors, the American Society of Clinical Pathologists (ASCP) Practice Assessment Committee, and the ASCP Board of Directors about the appropriate strategies for each clinical condition or laboratory test discussed in this parameter. 16 Am J Clin Pathol 2000;113:12 17 American Society of Clinical Pathologists

AJCP / SPECIAL ARTICLE This parameter is designed primarily as an educational resource for physicians in the provision of quality medical services. Adherence to this parameter is completely voluntary and does not necessarily assure a successful medical treatment or result. This practice parameter should not be considered inclusive of all proper procedures or tests that are reasonably directed to obtaining the same results. The physician should apply professional judgment to the unique clinical circumstances presented by the particular specific procedure or test. Physicians are encouraged to document the reasons for whatever procedure or test they use (whether or not in conformance with this parameter). Physicians should also take care to consider other medical and scientific advances that are available after the date of adoption of this parameter. The practice parameter was developed exclusively for the purposes set forth above and not for use in connection with matters involving reimbursement, credentialing, or utilization review. This practice parameter was reviewed by the members of the College of American Pathologists Transfusion Medicine Resource Committee. 3. Dienstag JL, Isselbacher KJ. Acute viral hepatitis. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison s Principles of Internal Medicine. 14th ed. New York, NY: McGraw-Hill; 1997:1677-1695. 4. Dienstag JL, Isselbacher KJ. Chronic hepatitis. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison s Principles of Internal Medicine. 14th ed. New York, NY: McGraw-Hill; 1997:1696-1704. 5. Lee WM. Hepatitis B virus infection. N Engl J Med. 1997;337:1733-1745. 6. Iwarson S, Norkrans G, Wejstal R. Hepatitis C: natural history of a unique infection. Clin Infect Dis. 1995;20:1361-1370. 7. Hu K-Q, Vierling JM. Molecular diagnostic techniques for viral hepatitis. Gastroenterol Clin North Am. 1994;23:479-498. 8. Purdy MA, Krawczynski K. Hepatitis E. Gastroenterol Clin North Am. 1994;23:537-546. 9. Alter HJ. The cloning and clinical implications of HGV and HGBV-C [editorial]. N Engl J Med. 1996;334:1536-1537. References 1. Noskin GA and the AMA Advisory Group on Prevention, Diagnosis, and Management of Viral Hepatitis. Prevention, diagnosis, and management of viral hepatitis: a guide for primary care physicians. Arch Fam Med. 1995;4:923-934. 2. Herrera JL. Serologic diagnosis of hepatitis. South Med J. 1994;87:677-684. American Society of Clinical Pathologists Am J Clin Pathol 2000;113:12 17 17