Viral Hepatitis Workup

Updated: Jun 12, 2017
  • Author: Naga Swetha Samji, MD; Chief Editor: BS Anand, MD  more...
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Workup

Approach Considerations

A simple screening test for the nonicteric patient with suspected viral hepatitis involves checking the urine for presence of bilirubin. As an alternative, a liver enzyme panel (generally a costly test) could be obtained. Bedside fingerstick glucose testing is important to evaluate for hypoglycemia in patients with an altered or questionable mental status.

Total bilirubin levels may be elevated in infectious hepatitis. Bilirubin levels higher than 30 mg/dL indicate more severe disease. Levels of alkaline phosphatase (ALP) are usually in the reference range but may elevate to no higher than twice the normal level. If ALP is elevated significantly, consider liver abscess or biliary obstruction.

A prolonged prothrombin time (PT), if present, is a grave finding indicating impaired synthetic function of the liver. Blood urea nitrogen (BUN) and serum creatinine levels should be assessed to look for evidence of renal impairment. Decreased renal function suggests fulminant hepatic disease. Serum ammonia should be measured in patients with altered mental status or other evidence of hepatic encephalopathy.

Detection of immunoglobulin M (IgM) for hepatitis A virus (HAV) is the standard for diagnosing acute infection with HAV.

Detection of IgM for hepatitis B core antigen (HBcAg) in serum is required to make the diagnosis of acute hepatitis B virus (HBV) infection. Hepatitis B surface antigen (HBsAg) may be present in acute infection or in patients who are chronic carriers. Its presence in patients with symptoms of acute hepatitis strongly suggests acute HBV infection but does not rule out chronic HBV with acute superinfection by another hepatitis virus. The presence of HBsAg in the serum for 6 months or longer indicates chronic infection.

Hepatitis C virus (HCV) infection can be confirmed with serologic assays to detect antibody to HCV (anti-HCV) or with molecular tests for the presence of viral particles. Third-generation assays for anti-HCV are sensitive and specific and can detect such antibodies within 4-10 weeks of infection. A rapid antibody test strip is available. Qualitative polymerase chain reaction (PCR) assay for presence of viral particles is the most specific test of HCV infection and may be helpful in diagnosing acute HCV infection before antibodies have developed.

Assays to detect IgM antibody to hepatitis D virus (HDV) do not need to be routinely performed in all patients with suspected hepatitis.

No specific imaging studies are required to make the diagnosis of hepatitis. However, obtain the appropriate diagnostic imaging studies (eg, ultrasonography or computed tomography [CT]) if the differential diagnosis favors gallbladder disease, biliary obstruction, or liver abscess.

Liver biopsy may be recommended for the initial assessment of disease severity in patients with chronic hepatitis B or chronic hepatitis C infection.

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Hepatitis A

Acute infection is documented by the presence of immunoglobulin M (IgM) antibody to hepatitis A virus (HAV) (anti-HAV), which disappears several months after the initial infection. The presence of immunoglobulin G (IgG) anti-HAV merely demonstrates that an individual has been infected with HAV at some point in the past, from 2 months ago to decades ago. IgG anti-HAV appears to offer patients lifelong immunity against recurrent HAV infection.

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Hepatitis B

Acute self-limited infection

Hepatitis B surface antigen (HBsAg) is the first serum marker seen in persons with acute infection. It represents the presence of hepatitis B virus (HBV) virions (Dane particles) in the blood. Hepatitis B e antigen (HBeAg), a marker of viral replication, is also present. When viral replication slows, HBeAg disappears, and antibody to HBeAg (anti-HBe) is detected. Anti-HBe may persist for years.

The first antibody to appear is antibody to hepatitis B core antigen (HBcAg) (anti-HBc). Initially, it is of the immunoglobulin M (IgM) class. Indeed, the presence of IgM anti-HBc is diagnostic for acute HBV infection. Weeks later, IgM anti-HBc disappears, and IgG anti-HBc is detected. Anti-HBc may be present for life. The anti-HBc (total) assay detects both IgM and IgG antibodies. The presence of anti-HBc (total) demonstrates that the patient has had a history of infection with HBV at some point in the past.

In patients who clear HBV, HBsAg usually disappears 4-6 months after infection, as titers of antibody to HBsAg (anti-HBs) become detectable. Anti-HBs is believed to be a neutralizing antibody, offering immunity to subsequent exposures to HBV. Anti-HBs may persist for the life of the patient.

Several key points should be kept in mind in interpreting serology findings from patients with acute HBV infection. The presence of HBsAg does not indicate whether the infection is acute or chronic. The presence of anti-HBc (IgM) is the sine qua non of acute HBV infection. The presence of anti-HBc (total) indicates that a patient has been infected with HBV at some point. The anti-HBc (total) remains positive both in patients who clear the virus and in patients with persistent infection.

The presence of anti-HBc (total) with a negative HBsAg and a negative anti-HBs indicates one of the following four things:

  1. The test result is a false positive

  2. The patient is in a window of acute hepatitis between the elimination of HBsAg and the appearance of anti-HBs; this scenario is not observed in patients with chronic HBV infection

  3. The patient has cleared HBV but has lost anti-HBs over the years

  4. The patient is one of the uncommon individuals with active HBV replication who is negative for HBsAg; this situation is diagnosed when either a positive HBeAg or a positive HBV DNA result is found

In some clinicians' opinions, the discovery of a lone positive anti-HBc (total) finding in the setting of negative HBsAg and negative anti-HBs findings mandates the performance of a polymerase chain reaction (PCR) assay for HBV DNA.

Chronic infection

HBsAg may remain detectable for life in many patients. Individuals who have positive findings for HBsAg are termed carriers of HBV. They may be inactive carriers or they may have chronic hepatitis. Anti-HBc is present in all patients with chronic HBV infections. HBeAg and HBV DNA may or may not be present. They reflect a state of active viral replication. HBV DNA levels are typically low or absent in inactive carriers. HBV DNA levels are higher in patients with chronic hepatitis B. High HBV DNA levels are associated with increased infectivity.

Anti-HBs is usually absent in patients with chronic infection. If anti-HBs is present in a patient who has positive HBsAg findings, it reflects the presence of a low level of antibody that was unsuccessful at inducing viral clearance.

Table 1 (below) summarizes diagnostic tests for HBV. [10, 13]

Table 1. Diagnostic Tests for Hepatitis B (Open Table in a new window)

Test

CHB HBeAg Positive

CHB HBeAg Negative

Inactive Carrier

HBsAg

+

+

+

Anti-HBs

-

-

-

HBeAg

+

-

-

Anti-HBe

-

+

+

Anti-HBc

+

+

+

IgM anti-HBc

-

-

-

HBV DNA

>2 × 104 IU/mL*

(>105 copies/mL)

>2 × 103 IU/mL

(>104 copies/mL)

3</sup> IU/mL

(<10<sup>4</sup> copies/mL)

ALT level

Elevated

Elevated

Normal

ALT = alanine aminotransferase; anti-HBc = antibody to hepatitis B core antigen; anti-HBe = antibody to HBeAg; anti-HBs = antibody to HBsAg; CHB = chronic hepatitis B; HBV = hepatitis B virus; HBeAg = hepatitis B e antigen; HBsAg = hepatitis B surface antigen; IgM = immunoglobulin M.

*Increasingly, experts in the field use IU/mL rather than copies/mL.

Markers after vaccination for HBV

The HBV vaccine delivers recombinant HBsAg to the patient without HBV DNA or other HBV-associated proteins. More than 90% of recipients develop protective anti-HBs. Vaccine recipients are not positive for anti-HBc unless they were previously infected with HBV.

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Hepatitis C

The tests most commonly used in the diagnosis of hepatitis C are liver chemistry, serology, hepatitis C virus (HCV) RNA testing, and liver biopsy.

In August 2012, the Centers for Disease Control and Prevention (CDC) expanded their existing, risk-based testing guidelines to recommend a one-time blood test for HCV infection in baby boomers—the generation born between 1945 and 1965, who account for approximately 75% of all chronic HCV infections in the United States—without prior ascertainment of HCV risk (see Recommendations for the Identification of Chronic Hepatitis C Virus Infection Among Persons Born During 1945–1965). [56] One-time HCV testing in this population could identify nearly 808,600 additional people with chronic infection. All individuals identified with HCV should be screened and/or managed for alcohol abuse, followed by referral to preventative and/or treatment services, as appropriate.

Liver chemistry

Elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels merely indicate the presence of liver injury. Patients with chronically elevated aminotransferase values should undergo a workup to exclude the possibility of chronic liver disease.

Measuring aminotransferase levels is an imperfect test in patients with documented HCV infection. The values do not predict the severity of clinical findings, the degree of histologic abnormalities, the patient’s prognosis, or the therapeutic response. Indeed, patients can have HCV-induced cirrhosis while still having normal liver chemistry values.

Although increases and decreases in aminotransferase levels do not appear to correlate with clinical changes, normalization of AST and ALT levels after acute infection may signal clearance of HCV. Normalization of AST and ALT levels while a patient is undergoing treatment with interferon predicts a virologic response to treatment. Similarly, an increase in AST and ALT values may signal a relapse after apparently successful drug therapy.

Serology

Structural and nonstructural regions of the HCV genome have been synthesized. These can be recognized by human immunoglobulin G (IgG) antibody to HCV (anti-HCV). Recombinant HCV antigens are used in enzyme-linked immunosorbent assay (ELISA) to detect anti-HCV in patients’ sera.

Anti-HCV test results remain negative for several months after acute HCV infection. Once anti-HCV appears, it usually remains present for the life of the patient—even in the 15% of cases in which the patient clears the virus and does not develop chronic hepatitis. Anti-HCV is not a protective antibody and does not guard against future exposures to HCV.

In 2010, the US Food and Drug Administration (FDA) approved the OraQuick HCV Rapid Antibody Test. [57]  It can be used for persons at risk for hepatitis or for those with signs or symptoms of hepatitis. The test strip can be used with a sample collected from oral fluid, whole blood, serum, or plasma.

Recombinant immunoblot assays (RIBAs) use recombinant HCV antigens that are fixed to a solid substrate. They are more specific than ELISA testing and have been used to confirm positive ELISA results. However, their use is being abandoned in favor of HCV RNA testing.

A positive HCV result with ELISA or RIBA has one of three potential interpretations, as follows:

  1. The test result is a true positive, and the patient is infected with HCV.

  2. The test result is a true positive, but the patient is no longer viremic for HCV and does not have chronic hepatitis; the results from neither ELISA nor RIBA distinguish resolved infection from active infection

  3. The test result is a false positive

ELISA testing has a positive predictive value (PPV) of more than 95% when used in patients at high risk for hepatitis C (eg, intravenous [IV] drug users and those with abnormal liver chemistry findings. However, its PPV is only 50-61% in patients at low risk for HCV infection. Furthermore, patients with autoimmune hepatitis or hypergammaglobulinemia frequently have false-positive ELISA results. Thus, a positive HCV ELISA or RIBA result does not prove that HCV infection is present. Positive serologic tests require confirmation with HCV RNA testing.

Other limitations of ELISA testing are that it fails to detect anti-HCV in 2-5% of infected patients and that it fails to detect anti-HCV in immunosuppressed patients (eg, patients with end-stage renal disease [ESRD], human immunodeficiency virus [HIV] infection, or concomitant immunosuppressant therapy). The possibility of HCV infection in this patient population should prompt HCV RNA testing.

HCV RNA testing

Since the early 1990s, polymerase chain reaction (PCR) assays and branched DNA assays have been used to detect HCV RNA in serum. In contrast to ELISA and RIBA testing, HCV RNA testing can confirm the presence of active HCV infection.

HCV RNA testing aids in the diagnosis of early cases of HCV infection (before the development of HCV antibody positivity or an elevated ALT level), seronegative cases (as in the setting of ESRD), and cases of perinatal transmission. It is also useful for confirming false-positive cases (eg, autoimmune hepatitis), assessing the HCV genotype and viral load, predicting the response to interferon therapy, guiding the duration and dose of interferon therapy, and determining the likelihood of relapse after a response to interferon therapy.

Liver biopsy

Liver biopsy is an important diagnostic test in possible cases of chronic hepatitis C. Biopsy results can help confirm the diagnosis, as well as help exclude other diseases that might have an impact on antiviral therapy, such as autoimmune hepatitis or hemochromatosis. Furthermore, liver biopsy offers the most reliable assessment of the severity of the disease.

Assessment of the degree of hepatic fibrosis is important for several reasons. The presence of advanced fibrosis (ie, stage 3 or 4) might trigger a decision to initiate screening to rule out the development of hepatocellular carcinoma (HCC) as a complication of advanced liver disease. Patients with previously unsuspected cirrhosis on biopsy should be monitored to ensure they do not develop large esophageal varices. Some clinicians consider that patients with stage 3 fibrosis should be regarded as “cirrhotic until proven otherwise.”

Knowledge of the severity of histologic changes may influence the patient and the physician to be either more or less aggressive in the pursuit of effective antiviral therapy. The presence of significant fibrosis (ie, stage 2, 3, or 4) might lead to a decision to initiate antiviral therapy in the hope that eradication of HCV would help to improve the patient’s long-term outcome. Patients with advanced histologic findings may seek experimental therapies should their condition not respond to standard antiviral therapy.

Patients with minimal fibrosis on biopsy (ie, stage 1 disease) might elect either to receive antiviral therapy or to postpone therapy. Indeed, the patient with stage 1 disease might be considered to be at low risk for complications of HCV infection. Furthermore, the risks of therapy might exceed benefits in such a patient (eg, a patient with HCV infection, stage 1 fibrosis and major depression).

In some clinicians' practices, before patients with stage 1 fibrosis elect to undergo a course of watchful waiting, they are advised that only virologic eradication of HCV can ensure that none of the extrahepatic complications of hepatitis C will develop. Patients are also advised to return for a repeat biopsy in 3-4 years to rule out progression of liver disease.

Liver biopsy has a number of noteworthy limitations. First, as an invasive procedure, it may be accompanied by significant complications (eg, bleeding) in approximately 1 in 1000 patients. Second, a sampling error may occur. Indeed, in some patients, the damage induced by viral infection is not uniform throughout the entire liver. In addition, interobserver variability in the assessment of histologic abnormalities may occur. Finally, as a snapshot in time, liver biopsy findings cannot be used to predict the rate of progression of chronic hepatitis C.

Other tests for estimating fibrosis in chronic hepatitis C

Liver stiffness can be estimated by using a technique known as vibration-controlled transient elastography or Fibroscan. The test is reportedly capable of diagnosing cirrhosis correctly in about 95% of patients; however it is less accurate in assessing patients with lesser degrees of fibrosis. [58] Fibroscan was approved for use in the United States in 2013 and, although it is not a replacement for liver biopsy, it can serve as a useful adjunct to help diagnose or exclude advanced fibrosis and cirrhosis. [59, 60]

Liver fibrosis can also be estimated by means of a number of commercial blood tests, including the following:

  • FIBROSpect II uses measurements of hyaluronic acid, tissue inhibitor of metalloproteinase-1 (TIMP-1) and alpha-2 macroglobulin to estimate liver fibrosis

  • HepaScore is based on levels of hyaluronic acid, alpha-2 macroglobulin, gamma glutamyl transferase (GGT), and total bilirubin, as well as age and sex

  • HCV FibroSURE measures alpha-2 macroglobulin, haptoglobin, GGT, bilirubin, ALT, and apolipoprotein A1

In general, these tests are considered accurate in determining the presence or absence of early (stage 1) or advanced (stage 4) fibrosis; however, they are considered to be less accurate in differentiating patients with moderate fibrosis.

At present, most gastroenterologists do not use serologic fibrosis markers as a substitute for liver biopsy. These tests may be useful for identifying patients at low risk for advanced disease (eg, asymptomatic women with HCV RNA positivity, persistently normal liver chemistry values, and no history of alcohol abuse or HIV infection) or for longitudinal follow-up of patients with minimal disease on biopsy who elect not to undergo antiviral therapy. If future generations of these markers achieve greater accuracy, they may obviate the need for liver biopsy.

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Hepatitis D and E

A serologic diagnosis of hepatitis delta virus (HDV) infection is made by using immunoglobulin M (IgM) antibody to HDV (anti-HDV) and IgG anti-HDV tests. IgM antibody to hepatitis B core antigen (anti-HBc) should be used to help distinguish between coinfection (positive for IgM anti-HBc) and superinfection (negative for IgM anti-HBc). Detecting HDV RNA in serum is also possible.

A serologic diagnosis of hepatitis E virus (HEV) infection is made by using IgM antibody to HEV (anti-HEV) and IgG anti-HEV. HEV RNA can be detected in the serum and stool of infected patients.

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Histologic Findings

Hepatitis B

Inactive carriers of hepatitis B virus (HBV) have no or minimal histologic abnormalities detected on liver biopsy specimens.

Patients with chronic hepatitis B may have a number of classic histologic abnormalities. Inflammatory infiltrates composed of mononuclear cells may either remain contained within the portal areas or disrupt the limiting plates of portal tracts, expanding into the liver lobule (interface hepatitis). Periportal fibrosis or bridging necrosis (between portal tracts) may be present. The presence of bridging necrosis places the patient at increased risk for progression to cirrhosis.

Ground-glass cells may be seen (see the image below). This term describes the granular homogeneous eosinophilic staining of cytoplasm caused by the presence of hepatitis B surface antigen (HBsAg). Sanded nuclei reflect the presence of an overload of hepatitis B core antigen (HBcAg). Special immunohistochemical stains may help detect HBsAg and HBcAg.

Liver biopsy specimen showing ground-glass appeara Liver biopsy specimen showing ground-glass appearance of hepatocytes in patient with hepatitis B.

Hepatitis C

Pathologists who interpret liver biopsy specimens frequently use a histologic scoring system introduced by Batts and Ludwig in 1995 (see Table 2 below) to grade hepatitis C virus (HCV)-induced disease. [61] The METAVIR scoring system developed by the French METAVIR Cooperative Study Group uses similar methodology.

Table 2. Histologic Grading for Hepatitis C–Induced Liver Disease (Open Table in a new window)

Grade

Portal Inflammation

Interface Hepatitis

Lobular Necrosis

1 – Minimal

Mild

Scant

None

2 – Mild

Mild

Mild

Scant

3 – Moderate

Moderate

Moderate

Spotty

4 – Severe

Marked

Marked

Confluent

Histologic staging for hepatitis C–induced liver disease is as follows:

  • Stage 1 – Portal fibrosis

  • Stage 2 – Periportal fibrosis

  • Stage 3 – Septal fibrosis

  • Stage 4 – Cirrhosis

Lymphocytic infiltrates, either contained within the portal tract or expanding out of the portal tract into the liver lobule (interface hepatitis), are commonly observed in patients with chronic hepatitis C. Portal and periportal fibrosis may be present. Other classic histologic features of the disease include bile duct damage, lymphoid follicles or aggregates, and macrovesicular steatosis.

Hepatitis D and E

The pathologic abnormalities associated with HBV-HDV coinfection are the same as those observed in patients infected with HBV alone (see above).

The classic pathologic findings associated with HEV infection include infiltration of portal tracts by lymphocytes and polymorphonuclear leukocytes, ballooned hepatocytes, acidophilic body formation, and intralobular necrosis of hepatocytes. Submassive and massive hepatic necrosis may be observed in severe cases.

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