Viral Hepatitis

Immunizations, blood supply testing, and safer injection practices have markedly decreased the incidence of new viral hepatitis infections, which in the U.S. have reached record low levels (1). Unfortunately, chronic infection remains common worldwide, and approximately one in 11 people are chronically infected with either hepatitis B (HBV) or hepatitis C (HCV). Because symptoms of chronic infection are minimal until late complications such as cirrhosis or hepatocellular carcinoma (HCC) develop, most chronically infected individuals are not aware of their infection.

Laboratory tests are key to recognizing exposure to and infection with hepatitis viruses to facilitate treatment and prevent complications (2). However, the many tests available for both HBV and HCV lead to much confusion among clinicians and laboratorians alike. This article reviews HBV and HCV tests, and their uses in diagnosis and in monitoring treatment.

Hepatitis B

HBV testing is complicated because of the many markers used in testing for infection, exposure, and immunity (Table 1). It is also unusual due to the virus's replication and life cycle. Briefly, once a liver cell becomes infected by the partially double-stranded DNA virus, viral DNA enters the nucleus and completes the second strand, forming covalently closed circular DNA (cccDNA). There are typically only a few copies of this per cell, which serve as the reservoir for production of RNA that codes for the HBV structural and non-structural proteins, as well as production of new viral particles.

The use of an RNA template creates some unique features. It leads to a high frequency of mutations that can affect laboratory tests and treatment response; however, it also allows use of reverse transcriptase inhibitors (RTI) to treat chronic HBV infection. Because RTIs prevent viral replication but do not in most cases affect cccDNA production, treatment is similar to that for HIV in that it improves patient health but does not cure infection.

The major means of acquiring HBV are through serum, including transfusion before testing was available, sexually, and by injection drug use. Children and babies acquire HBV easily through close contact with others who are infected, such as from mother to baby, and between children during early childhood. With greater than 2% prevalence in much of the world outside of North America and western and northern Europe, individuals born in high-prevalence areas are at high risk of HBV infection if not immunized as infants. In the United States, about 65% of those chronically infected are not aware of their infection, and most were born in endemic areas (2, 3).

Acute HBV

Acute HBV infection—though much less common—still occurs. In infants and young children, the infection is typically asymptomatic, but about one-third to one-half of older children and adults will develop jaundice. Elevated aspartate (AST) and alanine aminotransferase (ALT) activity, often in the 400–1000 U/L range, is the main laboratory feature.

At presentation, HBV surface antigen (HBsAg), HBV DNA, and total and immunoglobulin M antibody to HB core antigen (IgM anti-HBc) are typically present. HBV e antigen (HBeAg) may also be present, but is rarely tested. Usually absent are HBV surface antibody (anti-HBs) and HBV e antibody (anti-HBe). Textbooks refer to a core window in which IgM anti-HBc is positive, but HBsAg and anti-HBs are both negative. This core window phase usually occurs during recovery and not at presentation. With the HBsAg assays currently in use, none of the more than 200 acute HBV cases seen at our institution in the past 15 years were in the core window at presentation.

Most infected children and adults with an intact immune system will clear HBV within 6 months of presenting with acute infection, losing HBsAg, HBeAg, and HBV DNA, while developing both anti-HBs and anti-HBe—as well as retaining total anti-HBc. Patients typically lose HBsAg and HBV DNA within 1–3 months, and commonly are referred to as having resolved infection. Most still have cccDNA within hepatocytes, similar to what happens with herpes, cytomegalovirus, and varicella. As with those viruses, reactivation can occur with immunosuppression.

Chronic HBV

When HBsAg persists beyond 6 months, patients are considered to have chronic HBV infection. Most individuals will also have circulating HBV DNA, although in some the host immune response rapidly clears HBV viral particles from the circulation but not from the liver. At this stage, these patients are referred to as chronic carriers in the immune control phase. Chronic HBV develops in <5% of older children and adults, about 10–20% of immunosuppressed individuals, and in the majority of those infected before age 5—including >90% of those infected perinatally.

Most patients with chronic HBV acquired as older children and adults have evidence of ongoing liver injury, manifested by mild elevations in AST and ALT. However, many infected in the first 5 years of life will not have evidence of liver injury for many years, either by AST and ALT activity or on liver biopsy. This is the immune tolerant phase. Chronic HBV either may remain relatively asymptomatic, or have flares of activity that mimic acute hepatitis, including presence of IgM anti-HBc. These acute flares are often followed by loss of HBeAg or HBsAg, in about 5–7% and 0.5–1% of cases per year, respectively (4).

Historically, two different chronic HBV patterns have been recognized: HBeAg positive and HBeAg negative. When HBeAg testing came into use, viral load tests had high detection limits, often exceeding 5 x 106 copies/mL; most patients who were HBeAg positive had detectable viral loads, while HBeAg negative (and anti-HBe positive) individuals usually had undetectable viral loads. Using assays with detection limits <100 IU/mL, most HBeAg negative individuals have detectable viral load, and prognosis and treatment does not differ based on HBeAg status if viral load is similar.

Chronic HBV causes 80% of all primary liver cancers worldwide. The risk of complications such as cirrhosis and HCC is primarily related to three factors. For cirrhosis, the most important are the presence of ongoing liver injury—often identified by elevated ALT and AST—and viral load. For HCC, liver injury is not as important as viral load and genotype. Risk for both cirrhosis and HCC begins to increase at viral loads >104 copies/mL (2 x 103 IU/mL), and becomes very high at viral loads >107 copies/mL (2 x 106 IU/mL) (5, 6).

Table 1 Serologic and Virologic Markers for HBV
Viral Markers
Test	Significance	Uses
HBsAg (hepatitis B surface antigen)	Surface protein of HBV; circulates as part of viral particles, but mainly as free antigen.	Detect current infection with HBV; where available, quantitative HBsAg predicts success of treatment in eradicating virus.
HBeAg (hepatitis B e antigen)	Variant of HBV core antigen; produced by some, not all, HBV infections, circulates as free antigen, not part of viral particle.	Correlates with viremia in untreated patients (higher in HBeAg positive); during treatment, loss (and development of anti-HBe) predicts ability to discontinue therapy.
HBV DNA	Viral nucleic acid	Detect replicating virus; in treatment, used to monitor treatment efficacy.
HBV Resistance Mutations	Detects genetic differences in virus that confer resistance to reverse transcriptase inhibitors; some assays also detect mutations associated with loss of production of HBeAg.	Useful in patients previously exposed to reverse transcriptase inhibitors (either for treatment of HBV or HIV) to select appropriate treatment; occasionally in persons not responding to therapy for same reasons.
HBV Genotype	Detects strain of HBV infecting person.	Mainly used for research purposes, of little use clinically at present.
Viral Antibodies
Test	Significance	Uses
Anti-HBc	Antibody to HBV core antigen. First antibody to appear after exposure to virus; remains positive for longest with viral clearance. May be only marker many years after infection, especially in those also infected with HCV or HIV.	Total anti-HBc identifies exposure to HBV; IgM anti-HBc positive in acute HBV infection, but may be present with flares of activity in chronic HBV as well.
Anti-HBs	Antibody to surface antigen; protective against new infection with HBV. Develops with clearance of virus, and after immunization.	Detects immunity to HBV; often used to identify need for immunization in adults, or to prove response to HBV vaccine. If result <10 IU/mL, not considered protective.
Anti-HBe	Antibody to HBV e antigen. Develops both with viral clearance and in those who develop mutations that no longer code for production of HBeAg.	Usually positive if HBeAg negative, so of limited additional value. During treatment, if HBeAg becomes negative, appearance of anti-HBe usually indicates ability to discontinue treatment after additional 6–12 months.

HBV Treatment

The ultimate goal of treatment in HBV is clearance of HBV. Unfortunately, this goal is seldom met. Loss of circulating HBV DNA is, however, associated with lessened liver injury, improved liver histology, and reduced risk of cirrhosis or liver failure—and, perhaps, HCC. Loss of circulating HBV DNA also usually coincides with normalized ALT and AST levels. The two most widely used drugs, entecavir and tenofovir, have very low risk of resistant mutations, and high likelihood of loss of circulating HBV DNA (<20 IU/mL) (4). In patients who are HBeAg positive before treatment, loss of HBeAg and development of anti-HBe often is associated with long-term undetectable HBV DNA even off treatment, but this occurs in fewer than half. Those who are HBeAg negative before treatment or who remain HBeAg positive typically remain on treatment indefinitely. Quantitative HBsAg assays—not available in the U.S.—have been found to quickly predict likelihood of viral clearance (7).

HBV Reactivation

Immune suppression, either by medications or disease, often brings with it the return of viral replication in those who have cleared HBsAg or HBV DNA. Return of immune function is often associated with severe acute hepatitis and significant risk of death in these critically ill patients. Guidelines recommend testing for HBsAg and anti-HBc in persons who are immune-compromised or who will be treated with drugs causing immunosuppression (8). In our institution, depending on severity of immunosuppression, we either treat such individuals as we would those with chronic HBV to control viral replication or closely monitor them by measuring HBV DNA and HBsAg frequently, and treating if there is any increase.

HBV Testing and Interpretation

Many physicians have trouble interpreting HBV test results. Table 2 outlines the common patterns of HBV tests, as well as their significance and possible interpretation.

Table 2
Interpretation of Patterns of HBV Tests

HBs Ag	Anti- HBs	Total Anti- HBc	IgM Anti- HBc	HBe Ag	Anti- HBe	HBV DNA	Interpre- tation	Further testing suggested?
Neg	Neg	Neg	NP	NP	NP	NP	Never exposed to HBV	None unless further risk factors develop
Neg	Pos	Neg	NP	NP	NP	NP	Previous exposure to HBV, virus cleared	None unless immunosuppressed; in that case, either repeat along with HBV DNA regularly or consider treatment to prevent reactivation
Neg	Neg	Pos	NP or Neg	NP	NP	NP	Unclear; may be false positive anti-HBc or indicate remote exposure, especially if HCV or HIV co-infected	If HCV or HIV infected, consider exposed to HBV in past. If not, could perform anti-HBe, and if Pos, consider HBV exposed. Some suggest giving one dose HBV vaccine and checking anti-HBs after 1–2 weeks; if now Pos, consider HBV exposed. If immune suppressed, repeat HBsAg and HBV DNA regularly or consider treatment to prevent reactivation
Pos	Neg	Pos	Neg	Pos	Neg	Detect -ed	Chronic HBV, HBeAg positive	Consider for treatment; if treated, monitor HBV DNA, HBeAg, and anti-HBe regularly for effect
Pos	Neg	Pos	Neg	Pos	Neg	ND	Chronic HBV, HBeAg positive, likely on treatment	Continue to monitor HBV DNA, HBeAg, and HBe regularly for effect; if not on treatment, repeat HBV DNA (likely false negative)
Pos	Neg	Pos	Pos	NP, Pos, Neg	NP, Pos, Neg	NP, Pos, ND	Likely acute HBV; might also be chronic HBV during flair	Monitor HBsAg, anti-HBs at 3, 6 months to evaluate for clearance
Pos	Neg	Pos	Neg	Neg	Pos	Pos	Chronic HBV, HBeAg negative	Evaluate for treatment; if treated, repeat HBV DNA regularly for effect
Pos	Neg	Pos	Neg	Neg	Pos	Pos	Chronic HBV, HBeAg negative	Evaluate for treatment; if treated, repeat HBV DNA regularly for effect
Pos	Neg	Pos	Neg	Neg	Pos	ND	Chronic HBV, HBeAg neg, on treatment HBV carrier state (HBV infection with immune system control)	If on treatment, monitor HBV DNA regularly for effect; if not on treatment, may repeat HBsAg, HBV DNA periodically for spontaneous clearance or reactivation. If immunosuppressed, consider for treatment
Pos	Pos	Neg	Neg	NP	NP	NP	Uncommon occurrence; may be false-positive HBsAg or mutation in HBsAg	Refer to lab with special expertise for further testing
Pos	Neg	Neg	NP	NP	NP	NP	Uncommon occurrence; may be early HBV infection or false-positive HBsAg	If low signal-to-cutoff ratio for HBsAg, consider neutralization; if strongly positive, consider HBV DNA to detect early HBV infection, repeat in 1–2 months
Neg	Neg	Neg	NP	NP	NP	Pos	Uncommon occurrence; may be very early HBV infection or sample mix-up	Repeat studies in 1–2 months

Neg – Negative; Pos – Positive; NP – not performed; ND – not detected

Hepatitis C

From a laboratory standpoint, the diagnostic workup for HCV is simpler than for HBV, as there are only two tests for HCV markers, HCV viral load and HCV genotype, along with antibody to HCV. The more challenging areas with HCV testing involve false positive anti-HCV results and measuring HCV RNA during treatment.

Who Should Be Tested for HCV?

Traditionally, HCV testing has been based on risk factors, including blood transfusion before 1992, injection or snorting of drugs, or multiple sexual partners. Many infected individuals either do not admit or are not asked about these factors, and more than half of those infected are unaware of their disease (2). In the U.S., the overwhelming majority acquired infection during the peak of drug experimentation, so most were born between 1945 and 1965. Both the Centers for Disease Control and Prevention (CDC) and the U.S. Preventive Services Task Force recommend one-time screening of individuals born in that era, along with those who admit to risk factors (9, 10).

False Positive anti-HCV

Anti-HCV assays were developed to test blood donors, with the cutoff for classifying a result as positive established to assure maximum sensitivity. This has resulted in a higher number of false-positive results. In its 2003 guidelines for testing, CDC recommended confirming all results that were reactive but with a signal-to-cutoff ratio below that determined to have a high likelihood of being true positive (11). The CDC viral hepatitis website lists these values for most assays currently available (www.cdc.gov/hepatitis/HCV/LabTesting.htm, accessed 8/30/13).

Until 2012, the recommended confirmatory test was Recombinant Immunoblot Assay (RIBA), but its manufacture was temporarily halted in early 2012 and then discontinued in 2013. While similar confirmatory assays are available in other parts of the world, none are FDA-approved. The newest CDC guidance recommends confirming all reactive results with HCV RNA to identify currently infected individuals who may benefit from treatment (12).

There may be adverse consequences, however, from definitively labeling as positive patients who have false-positive results based on anti-HCV testing, even if they are HCV RNA-negative. In order to identify false positive results, CDC suggests repeating the anti-HCV test using a different manufacturer's assay, since false positive results tend to be assay specific (12).

In our laboratory, we routinely performed RIBA on 436 weakly positive results; 64.2% were RIBA-negative and 64.9% of 118 were also negative by a different manufacturer's assay. In a sample of 166 weakly positive results when RIBA was no longer available, a similar 64.6% were negative by a different manufacturer's assay.

Acute HCV

Although the incidence of acute HCV is increasing, diagnosis of this infection is difficult. Jaundice is rare, anti-HCV is negative at time of presentation in about half of cases, and there is no IgM anti-HCV test available. Diagnosis often is based on transient presence of HCV RNA, rising signal-to-cutoff ratio of anti-HCV at the time of recovery, or initial positive HCV RNA with negative anti-HCV.

HCV Treatment

Although details of HCV treatment are beyond the scope of this article, several treatment issues are relevant to laboratorians. In contrast to HBV, HCV treatment typically lasts for a defined period, with a goal of clearing infection. As with HBV, successful treatment is associated with improved liver histology, normalized AST and ALT levels, and better health outcomes. Successful treatment is defined as loss of virus on treatment, and continued absence of viremia 3–6 months after completing treatment, termed sustained virologic response (SVR). Most studies have found that patients who achieve SVR remain free of virus long-term in more than 99% of cases.

Clinicians base decisions on type and duration of treatment on several factors, the most important of which is genotype. As such, assays to determine genotype must be performed prior to treatment. Genotype 1 is the most common in Europe and North America, but has the poorest response to pegylated (long-lasting) interferon and ribavirin (PR). Since 2011, one of two drugs that directly target genotype 1 HCV protease have been added to PR, with SVR rates approaching those seen in other genotypes treated with PR.

Some of the primary factors that affect treatment response include viral load, degree of scarring in the liver, and, for treatment with PR plus a protease inhibitor, response to prior treatment with PR. In the case of genotype 1, an inherited trait based on mutations near the gene for IL-28b correlates with response to PR, but whether it provides much additional information in persons treated with PR plus a protease inhibitor is unclear (13). A number of other drugs with action against HCV have shown high activity in clinical trials, and some of these have now been FDA approved, expanding the number of treatment options (14).

Regardless of treatment, the rate and degree of viral response are important predictors of efficacy. While exact criteria and timing of measurement differ depending on various drug combinations, multiple measurements of HCV RNA early in therapy are important in determining both the likelihood of achieving SVR and, for combinations other than PR, whether to continue treatment. In PR therapy treatment failure is common, but the drugs do not appear to induce resistant mutations. In contrast, viral mutations that occur during treatment with protease inhibitors confer resistance. For these drugs, failure to clear the virus rapidly has been found a highly accurate indicator of likelihood of resistant mutations. Thus, if viral load is above a specific threshold for each protease inhibitor soon after treatment begins, treatment is stopped. Conversely, if no virus is detectable after 4 weeks of starting a protease inhibitor, most patients can undergo a shorter course of therapy.

The main issues for laboratories monitoring treatment are to provide HCV RNA assays with a very low detection limit and to make results available within a short period of time from sample collection. The latter is particularly important in enabling treatment to be stopped promptly if it is not working in order to keep resistant mutations from arising. For HCV RNA assays, guidelines suggest 10–15 IU/mL for detection limit and 25 IU/mL for quantification limit (15). Laboratories should report samples with detectable viral load below the limit of quantitation as target detected (16). Treatment response is poorer in those with detectable, but not quantifiable, HCV RNA (17). Those detection limits are typically only achievable using real time PCR. In our institution, our treating physicians expect an average turnaround time of 4 days, and 6 days maximum.

Summary

Despite advances in treatment and testing, HBV and HCV remain as significant challenges, and rely on laboratory tests for both diagnosis and treatment monitoring. Laboratorians should work closely with experts in this area to design appropriate testing panels for initial diagnosis and to assure adequate turnaround time for assays used to monitor therapy.

References

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