Volume 39, Supplement 1, Pages 31-35 (2003)

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Hepatitis B virus (HBV) DNA assays (methods and practical use) and viral kinetics

Article Outline

• 1. Introduction

• 2. HBV DNA, a marker of viral replication

• 3. Available HBV DNA tests

• 4. Practical use of HBV DNA quantification

• 4.1. Diagnosis of HBV infection

• 4.1.1. Acute hepatitis B

• 4.1.2. Chronic HBV infection

• 4.2. Assessment of disease severity and prognosis

• 4.3. Treatment of HBV infection

• 4.3.1. Decision to treat

• 4.3.2. Selection of optimal therapy

• 4.3.3. Treatment monitoring

• 5. HBV viral kinetics

• 5.1. HBV kinetics during acute infection

• 5.2. HBV kinetics during chronic infection

• 5.3. HBV kinetics during antiviral therapy

• 6. Conclusion

• References

• Copyright

1. Introduction

Virological diagnosis and monitoring of hepatitis B virus (HBV) infection are based on serologic assays detecting specific anti-HBV antibodies, and assays that can detect, quantify or characterize the components of HBV viral particles, such as HBV DNA and various viral antigens. HBV DNA detection and quantification now play a key role in the diagnosis of infection, therapeutic decision-making, and assessment of the response to therapy. However, clinically relevant HBV DNA thresholds remain to be established in various settings. HBV DNA quantification can also be used to monitor viral replication kinetics to better understand the mechanisms of infection and the virologic response to antiviral therapy.

2. HBV DNA, a marker of viral replication

The presence of HBV DNA in peripheral blood is a reliable marker of active HBV replication. HBV DNA is detectable within a few days after infection. It generally increases to reach a peak at the time of acute hepatitis, before progressively decreasing and disappearing when the infection resolves spontaneously [1]. In the patients progressing towards chronic hepatitis B surface antigen (HBsAg) carriage, chronic infection evolves through successive phases. HBV DNA levels are not stable over time and depend on the infection phase: the immuno-tolerance after acute infection is characterized by high levels of viral replication; the immuno-elimination phase is characterized by generally lower, often fluctuating HBV DNA; the ‘clinical latency’ phase is characterized by very low or undetectable levels of viral replication, depending on the sensitivity of the assay used; during reactivation phases, that are facilitated by immunosuppressive treatments, viral replication generally reaches high levels. During all phases in chronic HBsAg carriers, HBV DNA and supercoiled DNA (cccDNA, the persistent form of HBV in hepatocytes) are detectable in the liver with sensitive techniques. The HBV DNA load does not appear to be affected by the severity of liver lesions, but a non-causal relationship may exist, because liver lesions develop principally during the immuno-elimination phase.

3. Available HBV DNA tests

The principles of the molecular biology-based techniques that can be used to detect and quantify HBV DNA in peripheral blood have been reviewed recently [2]. These techniques are based on signal amplification following molecular hybridization (including the ‘hybrid capture’ and the ‘branched DNA’ methods) or target amplification (including polymerase chain reaction, PCR, and transcription-mediated amplification, TMA) [2]. Table 1 shows the commercial assays that can currently be used to detect and quantify HBV DNA. HBV DNA quantitative units used in the various assays do not represent the same actual amount of HBV DNA in a given clinical sample. The World Health Organization has established an international standard for universal standardization of HBV DNA quantification units, and an HBV DNA international unit (IU) has been defined [3]. International unit conversion factors for the non-standardized units used in commercial HBV DNA quantitative assays are currently being calculated. This IU must be preferred to any other quantitative unit and should now be implemented in all commercial HBV DNA quantitative assays for establishing clinically relevant thresholds and recommendations for clinical decisions based on HBV DNA load.

Table 1.

Available HBV DNA detection and quantification assaysa


Manufacturer	Assay	Method	Dynamic range of quantification
Digene Corp., Gaithersburg, MD, USA	HBV Digene Hybrid-Capture™ I	Hybrid capture signal amplification in tubes	700,000–560,000,000 copies/ml
	HBV Digene Hybrid-Capture™ II	Hybrid capture signal amplification in microplates	142,000–1,700,000,000 copies/ml
	Ultra-sensitive HBV Digene Hybrid-Capture™ II	Hybrid capture signal amplification in microplates after centrifugation	4700–57,000,000 copies/ml
Roche Molecular Systems, Pleasanton, CA, USA	Amplicor HBV Monitor™	Manual quantitative RT-PCR	1000–4,000,000 copies/ml
	Cobas Amplicor HBV Monitor™	Semi-automated quantitative RT-PCR	200–200,000 copies/ml
Bayer Corporation, Tarrytown, NY, USA	Versant™ HBV DNA 1.0 Assay	Manual branched DNA signal amplification	700,000–5,000,000,000 genome equivalents/ml
	Versant™ HBV DNA 3.0 Assay	Semi-automated branched DNA signal amplification	2000–100,000,000 copies/ml or 350–17,850,000ui/ml

The dynamic ranges of quantification are given in non-standardized units. Conversion factors to IUs are currently being calculated.

The dynamic ranges of quantification of the available assays are shown in Table 1 (in non-standardized units). Samples with a viral content above the upper limit of a given assay must be retested after 1/10 or 1/100 dilution for accurate quantification. These assays have been shown to be specific and accurate within their respective dynamic ranges of quantification [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The possible influence of the HBV genotype on quantification has not been studied. Whatever the assay, differences or variations of less than 0.5 log (i.e. less than 3-fold) should not be taken into account, as they may be due to intrinsic or between-patient variability.

4. Practical use of HBV DNA quantification

4.1. Diagnosis of HBV infection

4.1.1. Acute hepatitis B

HBV DNA detection or quantification is not necessary for the diagnosis of acute hepatitis B, which is based on serologic testing.

4.1.2. Chronic HBV infection

In chronic HBV infection, HBV DNA detection-quantification is necessary to determine whether or not HBV is replicating [14]. The quantitative result does not really matter in the presence of hepatitis B ‘e’ antigen (HBeAg), because the diagnosis of replicating chronic hepatitis B can be made independent of the viral load. In contrast, the interpretation of HBV DNA quantification is difficult in HBeAg-negative/anti-HBe antibody-positive patients. Indeed, the ‘inactive carriers’ appear to have lower mean HBV DNA levels than the patients with clinically active chronic hepatitis B [15], [16]. However, the discriminating HBV DNA threshold remains to be established in appropriate clinical studies using highly sensitive and accurate HBV DNA assays, and standardized IUs [17].

4.2. Assessment of disease severity and prognosis

HBV DNA detection provides valuable prognostic information. Indeed, the presence of HBV DNA is associated with a significant risk of progression of chronic hepatitis B to cirrhosis and hepatocellular carcinoma [14]. This risk is low in the absence of detectable HBV DNA, except in patients with cirrhosis who may subsequently develop hepatocellular carcinoma despite the absence of HBV replication. The possible prognostic significance of HBV DNA load, i.e. what level of HBV DNA is associated with progressive liver disease, remains to be determined in appropriate clinical studies.

4.3. Treatment of HBV infection

4.3.1. Decision to treat

The decision to treat chronic hepatitis B must be made in patients with elevated serum alanine aminotransferase (ALT) activity, a liver biopsy showing chronic hepatitis with or without cirrhosis, and the presence of significant levels of HBV DNA. The decision to treat is practically easy if HBeAg is present. It is more difficult in HBeAg-negative patients with detectable HBV DNA and mild to moderate lesions on liver biopsy, because no precise clinically relevant HBV DNA thresholds are known. Prospective trials are needed to determine HBV DNA loads (in IU/ml) above which patients with chronic hepatitis B should be treated (and below which they should not).

4.3.2. Selection of optimal therapy

The current treatment of chronic hepatitis B is based on standard interferon (IFN)-α or lamivudine [18], [19], [20], [21], [22], [23]. The choice will change in the near future for the following reasons: promising preliminary results have been reported with pegylated IFN-α [24], [25]; new potent antiviral molecules, such as adefovir dipivoxil and others, are and will soon be available [26], [27], [28]; combination therapy has not been appropriately evaluated to date and might yield interesting results. HBV DNA quantification could help selecting optimal therapy. The patients with a low HBV DNA may have a higher rate of sustained response to IFN-α than those with a high HBV DNA level. Conversely, the patients with a high HBV DNA level might be the best candidates for antiviral therapy with nucleoside/nucleotide analogs. Again, the precise HBV DNA cutoff that discriminates between ‘low’ and ‘high’ pretreatment replication needs to be determined in prospective clinical trials, using standardized quantification units.

4.3.3. Treatment monitoring

HBV DNA quantification, together with repeated ALT determinations and HBeAg/anti-HBe antibody assessments in HBeAg-positive patients, is critical in treatment monitoring [18], [19], [20], [21], [22], [23]. Non-responders to IFN-α-based treatment have little or no change in HBV DNA load during therapy, whereas responders show a significant decrease. Successful IFN-α treatment is characterized by HBe seroconversion in HBeAg-positive patients, and a reduction in HBV DNA load below the detection cutoff of signal amplification assays. Small amounts of HBV DNA often remain detectable in HBe seroconverters with more sensitive target amplification assays. In patients receiving nucleoside/nucleotide analogs, the viral load significantly and rapidly decreases, but low-level replication remains detectable with sensitive assays in most cases. To what level HBV DNA should be reduced to ensure sustained virologic and clinical remission remains to be prospectively determined.

HBV resistance, which has been shown to be frequent with lamivudine monotherapy, is characterized by a relapse of HBV replication during treatment [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]. Patients with lamivudine resistance are generally kept on lamivudine or may be switched to adefovir dipivoxil in case of rapidly progressing liver disease.

5. HBV viral kinetics

The advent of sensitive, reproducible and accurate tools to measure HBV DNA levels has recently opened the way to the monitoring of HBV replication kinetics in various clinical settings.

5.1. HBV kinetics during acute infection

The kinetics of acute HBV infection have been recently characterized in patients infected from a single source [1]. HBV DNA was shown to replicate rapidly. The HBV DNA peak occurred on average 127±47 days after infection and reached very high levels, in the order of 1010 copies/ml. In these patients (who spontaneously recovered), HBV DNA clearance started at the time of symptomatic acute hepatitis. It followed a two or three phase decay pattern with an initial rapid decline with unclear mechanisms, and a final phase that might be related to infected hepatocyte loss. The peak HBV production rate was estimated to be on average in the order of 1013 virions per day, with a maximum production rate of each infected hepatocyte of 200–1000 virions per day only [1]. The authors estimated that at this peak rate of virion production, every possible single and most double mutations would be created each day [1]. The latter finding suggests that viral escape is not the primary mechanism of HBV persistence, which occurs in approximately 5% of adult patients with acute hepatitis B.

5.2. HBV kinetics during chronic infection

At the chronic stage of infection, HBV DNA kinetics are at a steady state, which explains that HBV DNA levels remain relatively stable over time within each phase of infection. The daily production rate of HBV virions has been estimated to be on average 1011 viral particles per day, with a mean half-life of free HCV virions in the order of 1 day [40], [41]. The minimum half-life of infected cells remains debated [40], [41].

5.3. HBV kinetics during antiviral therapy

HBV kinetics during antiviral therapy with various molecules have been recently described. Given the small number of reports and the small numbers of patients in each study, these results should be considered preliminary, pending further studies on larger groups of patients. In a Greek study, pegylated IFN-α 2b, 100 μg qw and 200 μg qw, induced a 29.4 and 67.4% mean inhibition of HBV replication (IFN-α blocking effectiveness), respectively. The addition of lamivudine to 100 μg qw of pegylated IFN-α 2b increased the mean blocking effectiveness to 90.7% [25]. The mean death rate of infected cells was increased with the higher dose of pegylated IFN-α and when lamivudine was added [25].

The administration of nucleoside/nucleotide analogs, such as lamivudine, adefovir dipivoxil or entecavir, was shown to be associated with a dose-dependent inhibition of HBV replication, over 95% on average with the usual dosages. Various patterns of response, including both bi- and triphasic patterns, have been reported, the significance of which remains unclear [42], [43], [44], [45], [46]. The second slope of viral decrease during lamivudine therapy was shown to be associated with baseline ALT levels in one study, emphasizing the relationship between disease activity and the immune response resulting in the death of infected cells [42]. The recent report of a sensitive and accurate method to quantify cccDNA in the liver [47] might improve interpretation of viral kinetics analyses in patients with chronic hepatitis B receiving antiviral therapy.

6. Conclusion

HBV DNA detection and quantification plays a key role in the management of patients with HBV infection. However, few practical guidelines can be derived at present because of the lack of published data and standardization of HBV DNA quantification units. Various clinical studies need to be performed in order to establish clinically relevant thresholds. A better understanding of the effects of antiviral drugs and the HBV DNA levels that should be targeted is also needed. The study of HBV kinetics might help to better understand the mechanisms of action of antiviral drugs and improve the global management of these patients.

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Department of Virology (EA 3489), Henri Mondor Hospital, University of Paris XII, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France

Tel.: +33-1-4981-2827; fax: +33-1-4981-4831

PII: S0168-8278(03)00136-3

doi:10.1016/S0168-8278(03)00136-3

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