HBV DNA quantification

Hybridization and/or signal amplification methods have been used to quantify HBV DNA for many years, and are of lower sensitivity. The development of more sensitive HBV quantification molecular assays (polymerase chain reaction [PCR]) revealed that HBeAg-negative patients may replicate HBV. In addition, a more recent technique based on ‘real time’ PCR technology has allowed accurate quantification over a broad range of HBV DNA levels. This in turn has made it possible to accurately identify HBV DNA values in the untreated patient, as well as to better monitor the antiviral efficacy of highly potent antiviral drugs. The current real-time PCR assays, however, raise some technical issues: for very high DNA levels above 7–8 logs the range of quantification may be insufficient, different genotypes may not be equally quantified, and HBV DNA levels lack standardized reporting units. Besides technical issues specific to any particular new technology, there are issues related in general to the use of HBV DNA assays in the assessment of HBV disease and monitoring of therapy. For example, precise thresholds to guide medical decisions need to be established; different therapies are associated with different endpoints in HBeAg-positive and -negative patients; response to therapy can be assessed by different means; ‘undetectable HBV DNA’ is difficult to standardize in clinical practice; and assays must be sensitive enough to detect a virologic breakthrough early enough to allow the decision to modify therapy, i.e. before the patient experiences an ALT flare. Once these issues are resolved, real-time PCR technology should become the standard technology for monitoring hepatitis B therapy.

HBV DNA sequence analysis

Various assays can be used to determine genotype, assess pre-core and core mutations, and to determine resistance mutations. At present, these tests are not yet routinely used in clinical practice.

The five phases of chronic hepatitis B infection

The first phase of chronic hepatitis B (immunotolerance) may last 2–4 weeks in healthy adults. In subjects infected at birth or in early childhood it may persist several decades.

In the immune tolerance phase spontaneous and treatment-induced HBeAg seroconversion (that is, loss of the HBeAg and production of HBeAb) is infrequent (<5% per year). HBV DNA levels are high, usually >20 000 IU/mL, transaminases are normal, and liver histology is benign. Prognosis is generally favorable for patients who are in this phase. A study of 240 patients in this phase from Taiwan (mean age 27.6 years) found that only 5% progressed to cirrhosis and none to HCC during a follow-up period of 10.5 years.⁴

The immune clearance phase (‘HbeAg-positive chronic hepatitis’) is characterized by flares of transaminases, believed to be manifestations of immune-mediated lysis of infected hepatocytes secondary to increased T-cell responses to hepatitis B core antigen (HBcAg) and HbeAg.^5,6 These flares may precede HBeAg seroconversion, but many flares only result in transient decreases in serum HBV DNA levels without loss of HbeAg. Some flares may lead to hepatic decompensation.^7,8 The duration of this phase and the frequency/severity of flares correlate with the risk of cirrhosis and HCC.⁹ So-called ‘seroconversion’ is the outcome of this phase; factors associated with higher rates of spontaneous HbeAg seroconversion include older age,¹⁰ higher transaminases levels,^11,12 and HBV genotypes (B > C).^13,14 In particular, genotype B is associated with a lower prevalence of HBeAg, HBeAg seroconversion at an earlier age, and more sustained virological and biochemical remission after HBeAg seroconversion.

In the inactive HBsAg carrier state prognosis is generally favorable, especially if this state is reached early. However, inactive cirrhosis may be observed in patients who had developed severe liver injury during the preceding ‘immune clearance’ phase. Some inactive carriers have reactivation of HBV replication (either spontaneously or as a result of immunosuppression). Reversion back to HBeAg positive status is also possible.

Most patients who are in the fourth phase (‘reactivation of HBV replication/HBeAg-negative chronic hepatitis B’) come from a state of inactive carrier lasting a variable period of time. However, some patients progress directly from HBeAg-positive chronic hepatitis to HBeAg-negative chronic hepatitis. This phase is characterized by a fluctuating course both in terms of transaminases levels and HBV DNA levels. HBV DNA levels are usually lower than in the HbeAg-positive patients but may reach 20 or 200 million IU/ml. Transaminases were reported intermittently normal in 44% of 164 HBeAg-negative/HBeAb-positive patients followed for 21 months.¹⁵

This finding is important because it implies that a single HBV DNA level determination and a single transaminases level are not sufficient to differentiate patients in the inactive HBsAg carrier state from those with HBeAg-negative chronic hepatitis. Given the fluctuating course of the fourth phase, serial testing is more reliable than a single test.¹⁶

Recent studies in Europe, Asia, and the United States have all reported an increased prevalence of HBeAg-negative and a decreased prevalence of HBeAg-positive chronic hepatitis.^17,18 This may be related to aging of existing carriers, decrease in new HBV infections, and increased awareness. This finding has important impact on treatment strategies.

The resolved phase of HBV infection is characterized by HBsAg seroclearance, which occurs at the rate of 0.5–1.0% per year in patients with chronic HBV infection.^10,19

Host factors

Exogenous factors

Viral factors

AGE AT INFECTION/OLDER AGE: A Taiwanese study reported significant correlation between incidence of cirrhosis and patient’s age at study entry (p < 0.001).²⁰ Peak age for symptomatic cirrhosis and HCC is 50–60 years.

MALE GENDER: Males are more susceptible to development of liver complications,²¹ and the male:female ratio for development of HCC is 5:1 in some populations.²² Crude mortality rate for HCC in Chinese males is 207 per 100 000 patient-years and 42.5 per 100 000 patient-years in females.

ALPHA FETOPROTEIN: A Taiwanese study reported cirrhosis to be more common in patients experiencing repeated episodes of severe acute exacerbations of chronic hepatitis B with AFP levels >100 ng/mL (57.1%, p < 0.001).²⁰

In a Hong Kong study, acute exacerbations of chronic hepatitis B with AFP >100 ng/mL were associated with cirrhosis complications and HCC.

NAFLD (nonalcoholic fatty liver disease): While there are no studies yet that address the potential effect of NAFLD on the progression of fibrosis in hepatitis B, this effect has been shown in hepatitis C.²⁵

ALCOHOL CONSUMPTION: Alcohol abuse increases the risk of HCC 2–4-fold relative to alcohol abstinence.²⁴ Heavy use of alcohol is also a risk factor for the development of cirrhosis in chronic hepatitis B. This corresponds to >20 grams/day in women and >30 grams/day in men.

COINFECTION: HIV is associated with more rapid onset of cirrhosis and incidence of HCC.¹⁰ HCV is associated with 2–6-fold increased risk of HCC in cirrhotic patients when compared to monoinfection.²⁴ HDV is associated with 3-fold increased risk of HCC in cirrhotic patients compared to monoinfection.²⁴

EXPOSURE TO AFLATOXIN: Aflatoxin is present in grains and in developing countries is known to contribute to the development of HCC. Some authors have proposed a synergism of action between aflatoxin and HBV in producing HCC.⁹⁶

GENOTYPE: Evidence is currently emerging for a role of genotype in the progression of chronic HBV hepatitis.²⁶ Genotype D is associated with more severe liver disease and higher incidence of HCC at a younger age (<40 years) than genotype A.^27–29 Conflicting data exist for genotypes B and C.^27–29

HBeAg/anti-HBe STATUS: There is conflicting evidence in regards to a possible association between HBeAg positivity and the risk of HCC. In a prospective study of 684 Taiwanese HBsAg-positive patients with histologically proven chronic hepatitis, those who were anti-HBe positive were equally at risk for the development of cirrhosis as those who were HBeAg positive.²¹ Another prospective Taiwanese study of >11 000 men³⁰ found that the relative risk of developing HCC was much higher for HBeAgpositive/HBsAg-positive men than for HBeAgnegative/HBsAg-positive men and higher than for HBeAg-negative/HBsAg-negative men. However, because HBeAg/HBeAb status was recorded only upon study entry, it is likely that during the long follow-up (10 years) a substantial proportion of the HBeAg-positive patients seroconverted to HBeAb before developing HCC. For this reason it is difficult to conclude that HBeAg positivity is associated with an increased risk of HCC.

HBsAg SEROCLEARANCE: This produces favorable biochemical, virological, and histologic parameters, but may not reduce the risk of HCC in patients when seroclearance occurs after the age of 50 years and/or after development of cirrhosis.^31–33

VIRAL LOAD: Prolonged low-level viremia is probably more influential in the development of cirrhosis and HCC than short-lived, high-level viremia.³¹ Recommendations of the American Association for the Study of Liver Diseases (AASLD) state that HBeAg-positive patients with serum HBV DNA levels above 20 000 IU/mL should be considered for treatment. However, there is evidence that active disease occurs at lower levels of HBV DNA, particularly in HBeAg-negative patients who have lower levels of HBV DNA than those with HBeAg-positive disease. In a Chinese retrospective study, 45% of HBeAg-negative patients with evidence of inflammation (raised ALT levels) had HBV DNA levels below 20 000 IU/mL.³⁴ These findings suggest that using a threshold value of HBV DNA 20 000 IU/mL to differentiate active and inactive disease in HBeAg-negative patients will exclude a substantial proportion of patients with active hepatitis. For this reason HBeAg-negative patients with lower HBV DNA levels (2 000–20 000 IU/mL) undergo liver biopsy and be treated if significant inflammation of fibrosis are present.

cccDNA (= COVALENTLY CLOSED CIRCULAR DNA): Infection of hepatocytes with HBV is followed by conversion of the relaxed circular viral DNA genome (rcDNA) into covalently closed circular DNA (cccDNA). cccDNA is localized to the nucleus, where it is transcribed to form the viral mRNA. cccDNA is an important pool of viral DNA that acts as a template for viral replication but is relatively resistant to existing therapies. Two Hong Kong studies have investigated the changes in cccDNA levels as chronic hepatitis B progresses.^31,35

One study quantified total HBV DNA and cccDNA levels in sera and liver biopsies from 16 HBeAgpositive patients and 36 anti-HBe-positive patients. Intrahepatic and serum HBV DNA and cccDNA levels were significantly lower in subjects with anti-HBe-positive disease. In the early replicative phase, during which total intrahepatic HBV DNA levels were high, only a small proportion was present in the form of cccDNA. As the disease progressed, total viral load decreased and cccDNA became the predominant form of intrahepatic HBV DNA. In the second study, on 92 patients who had undergone HBsAg clearance, intrahepatic total and cccDNA were qualitatively measured in 16 patients. Of these, 37.5% had detectable intrahepatic DNA, predominantly in the form of cccDNA. The results of these two studies demonstrate that as chronic hepatitis B progresses and viral load decreases, cccDNA becomes the principal form of HBV DNA in the liver. This represents a stable pool of intrahepatic viral DNA that has impacts for successful antiviral therapy and may explain the viral exacerbations often reported in anti-HBe-positive patients.

HBsAg-positive pregnant women

Vaccination breakthrough has been reported in newborns of mothers carrying HBV DNA >1.2 × 10⁹ copies/mL (N >2.4 × 10⁸ IU/mL). Lamivudine administered in the third trimester of pregnancy has been shown to be effective in reducing vertical transmission.³⁷ Although the use of prophylactic lamivudine in pregnant women to prevent maternal–fetal transmission is still a matter of controversy, these patients should be evaluated case by case in conjunction with a specialist.