The diagnosis of hepatitis B virus (HBV) infection was initiated by the discovery of the Australia antigen (Hepatitis B surface antigen, HBsAg). During the ensuing decades, serologic assays were established for HBsAg and other HBV antigens and antibodies. Advances in molecular biology techniques led to the development of polymerase chain reaction (PCR) assays for direct determination of hepatitis B virus DNA (HBV DNA).
Diagnosis of HBV infection tests for a series of serological markers of HBV and excludes alternative etiological agents such as hepatitis A, C, D, and E viruses. Serological tests are used to distinguish acute, self-limited infections from chronic HBV infections and to monitor vaccine-induced immunity. These tests are also performed to determine if the patient should be considered for antiviral therapy. Nucleic acid testing for HBV DNA is used as the standard to quantify HBV viral load and measures, together with HBV antigens and HBV antibodies, the effectiveness of therapeutic agents.
Other causes of chronic liver disease should be systematically looked for including coinfection with HCV, HDV, HEV or HIV. Cytomegalovirus, Epstein-Barr virus, enteroviruses, other hepatotoxic drugs, and even herbal medicines should be considered when appropriate. Moreover, comorbidities, including alcoholic, autoimmune and metabolic liver disease with steatosis or steatohepatitis should be assessed. Finally, vaccination status and previous test results should be used to guide appropriate testing.
Serological tests for viral antigens can be performed on either serum or plasma (Yang 2002). The World Health Organization (WHO) has defined an international standard for normalisation of expression of HBV DNA concentrations already long time ago (Quint 1990). Serum HBV DNA levels should be expressed in IU/mL to ensure comparability; the same assay should be used in the same patient to evaluate antiviral efficacy. Both HBV antigens and antibodies are stable at room temperature for days, at 4°C for months, and frozen at –20°C to –70°C for many years. Care should be taken to avoid hemolysis of the sample because it may interfere with the ability of the assay to accurately detect these markers. Vigilance must be taken to avoid the degradation of the viral nucleic acid in the specimen, which can result in falsely low or no measurable viral load. Serum should therefore be removed from clotted blood within 4 hours of collection and stored at –20°C to –70°C (Krayden 1998). Alternatively, the presence of EDTA in plasma is known to stabilise viral nucleic acids. EDTA blood can be stored for up to five days at 4°C without affecting the viral load. Polymerase chain reaction-based tests that are routinely used as standard can use either serum or plasma. In principle, the diagnosis of HBV infection can also be made by the detection of HBsAg or hepatitis B core antigen (HBcAg) in liver tissues by immunohistochemical staining.
Hepatitis B surface antigen (HBsAg) is the serologic hallmark of acute and chronic HBV infection. The HBsAg level is a reflection of the transcriptional activity of the matrix of HBV infection, the covalently closed circular HBV DNA (cccDNA). It is an important marker that not only indicates active hepatitis B infection but can also predict clinical and treatment outcomes. It can usually be detected by chemiluminescent microparticle immunoassay (CMIA) technology. Immunoassays are standardised against the WHO international standard, relatively inexpensive, fully automated and express HBsAg titres in IU/mL.
HBsAg appears in serum 1 to 10 weeks after acute exposure to HBV, prior to the onset of hepatitis and elevation of serum alanine aminotransferase. HBsAg usually becomes undetectable after four to six months in patients who recover from hepatitis B. Persistence of HBsAg for more than six months implies chronic infection. It is estimated that about 5 percent of immunocompetent adult patients with genuine acute hepatitis B progress to chronic infection (Chu 1989). Among patients with chronic HBV infection, the rate of clearance of HBsAg is approximately 0.5 to 1 percent per year (Liaw 1991). The disappearance of HBsAg is frequent, but not always followed by the appearance of hepatitis B surface antibody (anti-HBs). In most patients, anti-HBs persists for decades, thereby conferring long-term immunity. The coexistence of HBsAg and anti-HBs has been reported in HBsAg positive individuals (Tsang 1986, Dufour 2000). In most instances, the antibodies are unable to neutralise the circulating virions. These individuals should therefore be regarded as carriers of the hepatitis B virus.
In recent years the quantification of HBsAg levels (qHBsAg) has become more important. Assays for qHBsAg are fully automated and have high output. The two most reported assays for the quantification of serum HBsAg are the ARCHITECT HBsAg QT (Abbott Laboratories) and the Elecsys HBsAg II Quant (Roche Diagnostics). These assays detect all forms of circulating HBsAg: virion-associated as well as subviral filamentous and spherical particles (Bayliss 2013), possibly as well as HBsAg produced from integrated viral envelope DNA, which needs to be put into consideration in different clinical settings. qHBsAg titres are higher in HBeAg(+) than in HBeAg(-) patients and are negatively correlated with liver fibrosis in HBeAg(+) patients. In HBeAg(-) chronic hepatitis B, an HBsAg level <1000 IU/mL and an HBV DNA titre <2000 IU/mL accurately identifies inactive carriers (Brunetto 2010). During PEG-IFN treatment, HBsAg quantification is used as an on-treatment stopping rule to identify patients who will not benefit from therapy, and treatment may be stopped or switched at week 12 (EASL 2012). In contrast, in patients with nucleos(t)ide therapy the measurement of qHBsAg levels over time during antiviral therapy have not yielded definite answers yet in helping to distinguish patients that will clinically resolve chronic hepatitis B infection with HBsAg loss or seroconversion. Interestingly, stopping antiviral treatment in association with low HBsAg titres seems to be a new area of HBV related clinical research (Papatheodoridis 2016). In clinical practice, HBsAg quantification is a simple and reproducible tool that can be used in association with HBV DNA to classify patients during the natural history of HBV and to monitor therapy and the use of both parameters has been linked to the assessment of a ship´s “longitude and latitude” position in the ocean (Martinot-Peignoux 2013).
Since most HBsAg assays relay on elisa technique sophisticated laboratory equipment is needed to perform the assay. To allow screening in resource limited settings, rapid diagnostic test (RDT) have been developed for the detection of HBsAg. The latest assays e.g. VIKIA HBsAg, Alere Determine HBsAg or DRW-HBsAg v2 assay showed in recent studies high sensitivity and were able to detect HBsAg mutation variants and might present in the future powerful tools for screening campaigns (Servant-Delmas 2015).
Hepatitis B core antibody (Anti-HBc) can be detected throughout the course of HBV infection in the serum and it appears after HBsAg.
During acute infection, anti-HBc is predominantly class IgM, which is an important marker of HBV infection during the window period between the disappearance of HBsAg and the appearance of anti-HBs. IgM anti-HBc may remain detectable for up to two years after acute infection. Furthermore, the titre of IgM anti-HBc may increase to detectable levels during exacerbations of chronic hepatitis B (Maruyama 1994). This can present a diagnostic problem, incorrectly suggesting acute hepatitis B. Other common causes of acute exacerbation of chronic hepatitis B are superinfection with hepatitis D virus (delta virus) or hepatitis C virus. IgG anti-HBc persists along with anti-HBs in patients who recover from acute hepatitis B. It also persists in association with HBsAg in those who progress to chronic HBV infection.
Isolated detection of anti-HBc can occur in three settings: during the window period of acute hepatitis B after disappearance of HBsAg when the anti-HBc is predominantly IgM; many years after recovery from acute hepatitis B when anti-HBs has fallen to undetectable levels; and after many years of chronic HBV infection when the HBsAg titre has decreased to below the level of detection.
HBV DNA can be detected in the liver of most persons with isolated anti-HBc. Transmission of HBV infection has been reported from blood and organ donors with isolated anti-HBc. There are, in a small percentage of cases, false positive isolated anti-HBc test results.
The evaluation of individuals with isolated anti-HBc should include repeated testing for anti-HBc, HBsAg, anti-HBe, and anti-HBs. Those who remain isolated anti-HBc positive should be tested for the presence of IgM anti-HBc to rule out recent HBV infection. Individuals with evidence of chronic liver disease should be tested for HBV DNA to exclude low-level chronic HBV infection.
Hepatitis B e antigen (HBeAg) is a secretory protein processed from the precore protein. It is generally considered to be a marker of HBV replication and infectivity. HBeAg to anti-HBe seroconversion occurs early in patients with acute infection, prior to HBsAg to anti-HBs seroconversion. However, HBeAg seroconversion may be delayed for years to decades in patients with chronic HBV infection. In such patients, the presence of HBeAg is usually associated with the detection of high levels of HBV DNA in serum and active liver disease and is associated with higher rates of transmission of HBV infection. However, HBeAg positive patients with perinatally acquired HBV infection may have normal serum ALT concentrations and minimal inflammation in the liver (Chang 1988).
Seroconversion from HBeAg to anti-HBe is usually associated with a decrease in serum HBV DNA and remission of liver disease. However, some patients continue to have active liver disease after HBeAg seroconversion. Such individuals may have low levels of wild type HBV or HBV variants with a stop codon in the precore or dual nucleotide substitutions in the core promoter region that prevent or decrease the production of HBeAg (Carman 1989).
Qualitative and quantitative tests for HBV DNA in serum have been developed to assess HBV replication. Currently, most HBV DNA assays use real-time PCR techniques, report results in IU/mL, have a lower limit of detection of up to 9 IU/mL and a range of linearity of up to 9 log10 IU/mL.
Recovery from acute hepatitis B is usually accompanied by the disappearance of HBV DNA in serum. However, HBV DNA may remain detectable in serum for many years if tested by PCR assays (Cornberg 2011) suggesting that the replication machinery of the virus persists but is controlled by the immune system (occult infection with low amounts of HBV DNA in the absence of HBsAg).
In patients with spontaneous or treatment-induced HBeAg seroconversion in chronic hepatitis B, PCR assays may remain positive except in patients with HBsAg loss or seroconversion. By contrast, most patients who develop HBeAg seroconversion during nucleos(t)ide analogue therapy have undetectable serum HBV DNA. In fact, many patients receiving nucleos(t)ide analogue therapy remain HBeAg positive despite having undetectable serum HBV DNA for months or years. The explanation for this phenomenon is likely related to the lack of direct effect of nucleos(t)ide analogues on cccDNA and viral RNA transcription and viral protein expression.
HBV DNA levels are also detectable in patients with HBeAg negative chronic hepatitis, although levels are generally lower than in patients with HBeAg positive chronic hepatitis. Because of the fluctuations in HBV DNA levels there is no absolute single cutoff level that is reliable for differentiating patients in the inactive carrier state from those with HBeAg negative chronic hepatitis B (Chu 2002).
HBV can be classified actually into ten genotypes (A to J) and four major serotypes with approximately 4 and 8% intergroup nucleotide divergence across the complete genome.
Genotypes A-D, F, H, and I are classified further into subgenotypes (Kramvis 2014). There have been reports about differing therapeutic responses with nucleos(t)ide analogues and interferon α with respect to different genotypes giving a greater chance of therapeutic response with IFN in genotype A. Furthermore, some genotypes, such as B and C, may have a greater risk for the development of hepatocellular carcinomas. HBV genotyping can be determined using several methods; most diagnostic laboratories use commercial available line probe assays (e.g. Inno-Lipa®), or Sanger sequencing but other assays such as reverse hybridisation, restriction fragment length polymorphism (RFLP), -specific PCR assays, sequence analysis, microarray (DNAchip), real time PCR and fluorescence polarisation assay (Villar 2015) can be used. Nevertheless, in contrast to hepatitis C, the diagnosis of HBV genotypes in the clinical setting is not routine (Thursz 2011).
Hepatitis B virus (HBV) mutations associated with resistance to HBV drugs arise frequently, and these can sometimes lead to treatment failure and progression to advanced liver disease. Considerable research is focused into the mechanisms of resistance to nucleos(t)ides and the selection of mutants. The genes that encode the polymerase and envelope proteins of HBV overlap, so resistance mutations in the polymerase usually affect the hepatitis B surface antigen; these alterations affect infectivity, vaccine efficacy, pathogenesis of liver disease, and transmission throughout the population (see Chapter 2). Associations between HBV genotype and resistance phenotype have allowed cross-resistance profiles to be determined for many commonly detected mutants, so genotyping assays can be used to adapt therapy. In vitro phenotyping procedures are established in a rather small number of HBV laboratories and are not commercially available. Known mutations can be detected by commercially available tests (line probe assay e.g. Inno-Lipa®) with a threshold of about 5% or by Sanger sequencing of the viral polymerase gene with a threshold of about 20%. Determination of novel mutations remains for research-oriented labs with full-length sequencing methods or novel ultra-deep next generation sequencing techniques (NGS) that allow detection of mutants with threshold below 1%.
The so-called hepatitis B core-related antigen (HBcrAg) assay utilizes a mixture of monoclonal antibodies isolated from HBV core antigen – immunized mice to detect and quantify HBV core antigen (HBcAg), free HBeAg, HBeAg-antibody complex, and the 22kDa precore protein (p22cr). HBcrAg can be measured using a commercially available chemiluminescent enzyme immunoassay and the levels are quantified in U/ml (Lumipulse, Belgium). The assay´s measurement linear range spans from 3 to 7logU/ml but shows some sensitivity issues below 3log U/ml; samples above 7log U/ml need to be diluted. Several reports suggest that HBcrAg levels correlate with serum HBV-DNA in untreated patients with CHB and might be useful to differentiate HBeAg negative patients with active and inactive disease. A correlation between HBcrAg levels and the size of the intrahepatic cccDNA pool has been suggested in cohorts of patients with genotype B/C, either untreated or undergoing NA treatment. HBcrAg has been also shown to correlate with intrahepatic viral RNA levels in Asian patients treated with NAs (reviewed in Charre 2019). A recent study found that HBcrAg is strongly correlated with total HBV DNA, cccDNA and pgRNA levels both in HBeAg positive and HBeAg negative patients. CccDNA transcriptional activity, calculated by pgRNA/cccDNA ratio, was only correlated to HBcrAg, but not to qHBsAg in HBeAg negative patients, suggesting that HBcrAg might be a better surrogate marker of cccDNA transcriptional activity than qHBsAg. Patients who were negative for HBcrAg (<3log U/ml) showed less liver cccDNA and lower cccDNA activity than HBcrAg positive patients. Furthermore, several recent studies (mainly in Asian patients) could link high or increasing HBcrAg levels with an elevated risk for HCC development (Tada 2016, Honda 2016). With more studies needed ahead, HBcrAg could become useful in the evaluation of new antiviral therapies aiming at a functional cure of HBV infection either by directly or indirectly targeting the intrahepatic cccDNA pool (Testoni 2019).
Another interesting marker is the quantitative detection of HBV-RNAs in serum. HBV RNAs can be detected in patients’ sera at levels ranging between 0.1 and 1% of HBV-DNA levels in the absence of antiviral treatment. Several observations suggest a wide heterogeneity of circulating HBV RNA species that may vary depending on different stages of chronic HBV infection. Methods of quantification of serum HBV RNAs differ according to studies. There are two main strategies to detect and quantify HBV transcripts. One is based on 3´end amplification that amplifies the majority of HBV transcripts (except truncated RNAs) as they share the same 3´end. Quantification of circulating truncated RNAs by assays using a primer in the cryptic polyA signal site could reflect the transcriptional activity of viral integration. The second strategy is based on 5´amplification specifically targeting 3.5kb transcripts, pgRNA and and preC mRNA. For testing, low input volumes are needed; however, their sensitivity remains to be improved: currently, the LOD ranges from 1.85log copies/ml to 3.4 log copies/ml, and there is currently no direct standardization of HBV RNA quantification. (reviewd in Charre 2019). Several studies could show that encapsidated and enveloped HBV-RNA can be detected in serum of chronic HBV infected patients in high quantity (Wong 2016, van Bömmel 2015). Furthermore, HBV-RNA in serum is highly correlated with intrahepatic pregenomic RNA as a surrogate for active cccDNA in NA or pegylated IFN treated and untreated conditions (Giersch 2017). In line with this data, patients with favourable response during pegylated IFN or nucleos(t)ide analogue treatments (van Bömmel 2015, Jansen 2016) could be predicted by quantitative monitoring HBV-RNA in serum. Only few studies so far have shown a moderate correlation of RNAs with qHBsAg in pretreated patients, in HBeAg neg patients there was only a weak correlation (Liu 2019). The loss of serum HBV RNAs may reflect the transcription silencing of cccDNA and may be an indicator for safe withdrawal from antiviral treatment. Theoretically, the most promising area for clinical application of serum HBV RNAs might be the prediction of relapse and sustained response, especially HBsAg loss after treatment discontinuation, but further studies are needed: for example, to better understand how serum HBV RNAs, specifically pgRNA, is released from hepatocytes, in the virion or naked capsid? Is the egress mechanism different between HBV DNA containing particles and RNA containing particles? Taken together, these data indicate that HBV-RNA might become an important marker for the monitoring of intrahepatic replication during antiviral treatments in clinical practice in the near future.
Finally, higher anti-HBc levels detected by quantitative measurements of anti-HBcAg was hypothesised to reflect a stronger host-adaptive anti-HBV immunity (Yan 2013). Interestingly, recent studies (in Asian patients) could show that quantitative level of anti-HBc is a new additional predictor of pegylated IFN and nucleos(t)ide analogue treatments efficacy in HBeAg positive patients and might be used for pretreatment stratification (Fan 2016).
As a first step, the causal relationship between HBV infection and liver disease has to be established and an assessment of the severity of liver disease needs to be performed. Not all patients with chronic HBV infection have persistently elevated aminotransferases. Patients in the immune-tolerant phase have persistently normal ALT levels and a proportion of patients with HBeAg negative chronic HBV may have intermittently normal ALT levels. Therefore appropriate, longitudinal long-term follow-up is crucial.
The assessment of the severity of liver disease should include: biochemical markers, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), gammaglutamyl transpeptidase (GGT), alkaline phosphatase, prothrombin time and serum albumin, blood counts, and hepatic ultrasound. Usually, ALT levels are higher than AST. However, when the disease progresses to cirrhosis, the ratio may be reversed. A progressive decline in serum albumin concentrations and prolongation of the prothrombin time, often accompanied by a drop in platelet counts, are characteristically observed once cirrhosis has developed (EASL 2012).
The diagnosis of acute HBV is based upon the detection of HBsAg and IgM anti-HBc. During the initial phase of infection, markers of HBV replication, HBeAg and HBV DNA, are also present. Recovery is accompanied by the disappearance of HBV DNA, HBeAg to anti-HBe seroconversion, and subsequently HBsAg loss or seroconversion to anti-HBs.
The differential diagnosis of HBsAg positive acute hepatitis includes acute HBV, exacerbations of chronic HBV, reactivation of chronic HBV, superinfection of a hepatitis B carrier with hepatitis C or D virus (Tassopoulos 1987), and acute hepatitis due to drugs or other toxins in an HBV carrier.
Previous HBV infection is characterised by the presence of anti-HBs and/or IgG anti-HBc. Immunity to HBV infection after vaccination is indicated by the presence of anti-HBs only.
In rare cases, anti-HBc immunoglobulin M (IgM) may be the only HBV marker detected during the early reconvalescence or ‘window period’ when the HBsAg and anti-HBs tests are negative. Because current tests for HBsAg are very sensitive, an anti-HBc IgM that is typically positive with acute HBV infection is not generally required to diagnose active infection. Because some chronic HBV carriers remain anti-HBc IgM positive for years, epidemiological information is necessary to confirm that the infection is indeed acute. A negative anti-HBc IgM in the presence of a positive HBsAg suggests that the infection is likely chronic. For these reasons, routine testing for anti-HBc IgM is not generally recommended to screen for acutely infected patients.
Chronic HBV infection is defined by the continued presence of HBsAg in the blood for longer than six months. Additional tests for HBV replication, HBeAg and serum HBV DNA, should be performed to determine if the patient should be considered for antiviral therapy. In addition HDV coinfection needs to be ruled out by testing for anti-HDV. All patients with chronic HBV infection should be regularly monitored for progression of liver disease because HBV DNA and ALT levels vary during the course of infection. In addition, patients who are not candidates for treatment at the time of presentation may become candidates for treatment during follow-up.
HBeAg negative patients who have normal serum ALT and low (<2000 IU/mL) or undetectable HBV DNA are considered to be in an inactive carrier state. These patients generally have a good prognosis and antiviral treatment is not indicated. However, serial tests are necessary to accurately differentiate them from patients with HBeAg negative chronic hepatitis who have fluctuating ALT and/or HBV DNA levels (Lok 2007). Patients who are truly inactive carriers should continue to be monitored but at less frequent intervals. HBeAg negative patients with elevated serum ALT concentrations should be tested for serum HBV DNA to determine if the liver disease is related to persistent HBV replication.
Antibody to hepatitis B core protein
Antibody to hepatitis B surface protein
Once an individual has been diagnosed with chronic HBV infection, follow-up testing must be performed for alanine aminotransferase (ALT), a marker of liver cell inflammation. Repeat periodic testing is indicated because the ALT levels can fluctuate (e.g., from less than the upper limit of normal to intermittently or consistently elevated). Sustained and intermittent elevations in ALT beyond the upper limit of normal are indicative of hepatic inflammation and correlate with an increased risk of progressive liver disease. It must be noted that the normal ALT ranges are both age and sex dependent and, occasionally, individuals with severe liver disease may not manifest elevated ALT (Cornberg 2011, EASL 2012).
This is defined as the presence of detectable HBV DNA by PCR in patients who are negative for HBsAg. Most of these patients have very low or undetectable serum HBV DNA levels accounting for the failure to detect HBsAg. Infections with HBV variants that decrease HBsAg production or have mutations in the S gene with altered S epitopes evading detection in serology assays for HBsAg are uncommon. HBV DNA is often detected in the liver and transplantation of livers from these persons can result in de novo HBV infection (Margeridon-Thermet 2009).
Immunity to HBV is acquired from a resolved infection or from vaccination. The HBV vaccine has been shown to induce protective immunity in 90% to 95% of vaccinees. Most vaccinees will have protective levels of anti-HBs for 5 to 10 years after vaccination, although the exact duration of immunity remains undefined.
Liver biopsy is still the standard procedure for determining the degree of necroinflammation and fibrosis since hepatic morphology can assist the decision to start treatment. Biopsy is also useful for evaluating other possible causes of liver disease such as fatty liver disease. Although liver biopsy is an invasive procedure, the risk of severe complications is low. It is important that the size of the needle biopsy specimen be large enough to accurately assess the degree of liver injury and fibrosis. A liver biopsy is usually not required in patients with clinical evidence of cirrhosis or in those in whom treatment is indicated irrespective of the grade of activity or the stage of fibrosis.
There is growing interest in the use of noninvasive methods, including serum markers and transient elastography, to assess hepatic fibrosis to complement or avoid a liver biopsy. Transient elastography offers high diagnostic accuracy for the detection of cirrhosis, although the results may be confounded by severe inflammation associated with high ALT levels and the optimal cut-off of liver stiffness measurements for HBV varies among studies (Cornberg 2011, EASL 2012, Terrault 2016, see also Chapter 15).
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