Journal of Hepatology
Volume 36, Issue 4 , Pages 549-551, April 2002

Clinical utility in quantifying serum HBV DNA levels using PCR assays

Division of Gastroenterology, University of Michigan Medical Center, 3912 Taubman Center, Ann Arbor, MI 48109-0362, USA

See Article, pages 543–546

Article Outline

 

The evaluation of patients with hepatitis B virus (HBV) infection has evolved from serological to molecular diagnostic assays. Advances in molecular biology techniques in the early 1980s led to the development of hybridization assays for serum HBV DNA with detection limits of 106–107copies/ml. The introduction of polymerase chain reaction (PCR) assays in the late 1980s decreased the limit of HBV DNA detection to 102–103copies/ml. The availability of molecular diagnostic assays has improved our understanding of the pathogenesis and natural history of HBV infection and facilitated the monitoring of response to treatment. They also generated new questions and dilemmas. Patients with undetectable serum HBV DNA using hybridization assays were previously thought to have non-replicative infection. These patients were deemed not to require treatment and hepatic inflammation if present was attributed to other causes of liver disease.

Using hybridization assays, patients who have recovered from acute HBV infection invariably have undetectable HBV DNA in serum. However, recent studies using more sensitive PCR assays found that low levels of HBV DNA, generally <103copies/ml [1], [2], may persist for many years after recovery from acute HBV infection [3]. Studies using PCR assays also found that the vast majority of patients with chronic HBV infection including those who are hepatitis B e antigen (HBeAg) negative, hepatitis B e antibody (anti-HBe) positive have detectable HBV DNA in serum [1], [2], [4], [5], [6], [7], [8], [9]. These findings raise a number of clinically important questions. First, what level of serum HBV DNA is associated with progressive liver disease? Second, at what level of serum HBV DNA is treatment indicated? Third, to what level should serum HBV DNA be reduced during treatment in order to ensure sustained virologic response, HBeAg seroconversion, and remission of liver disease?

The National Institutes of Health (NIH) Workshop on ‘Management of Hepatitis B’ held in September 2000 attempted to address some of these questions [10]. Consensus definition and diagnostic criteria for clinical terms relating to HBV infection were discussed. An arbitrary serum HBV DNA level of 105copies/ml was proposed to differentiate chronic hepatitis B from inactive carrier state. This level was chosen to include serum HBV DNA levels that would be detected by all commercial as well as in house non-PCR based assays. Studies in the 1980s and early 1990s before PCR assays became popular demonstrated that most patients who developed spontaneous or treatment induced HBeAg seroconversion with undetectable serum HBV DNA had normal aminotransferase (ALT) levels, reduced histologic activity, decreased risks of hepatic decompensation, and improved survival. The majority of these patients would have detectable HBV DNA if they had been tested by PCR assays. Thus, it seems that low serum HBV DNA levels may not necessarily be pathogenic and may not require treatment. However, the selection of 105copies/ml as a cut-off value for differentiating chronic hepatitis B (HBeAg positive as well as HBeAg negative) from inactive carrier state has not been validated. The threshold HBV DNA level that is associated with progressive liver disease is not known and may be dependent on host factors such as immune response, viral factors such as HBV genotype and mutations in the core promoter and precore regions, and environmental factors such as alcohol consumption. In addition, many patients with chronic HBV infection have fluctuating HBV DNA levels. Finally, assays for HBV DNA quantification are not well standardized.

It is generally accepted that HBV is not a cytopathic virus and that liver disease associated with HBV infection is mainly immune-mediated. Evidence that HBV is not directly cytopathic is most obvious in children and adolescents with perinatally acquired HBV infection, in whom HBeAg and high serum HBV DNA levels are present, but ALT levels and liver histology are usually normal. Indeed, several studies found that HBeAg positive patients with normal ALT have higher serum HBV DNA levels than those with elevated ALT [4], [9], [11]. Nevertheless, presence of HBV is needed for ongoing necroinflammation.

Several studies have attempted to examine the range of serum HBV DNA levels in HBeAg positive as well as in HBeAg negative patients and to determine if there is a relation between serum HBV DNA levels and liver disease. Serum HBV DNA levels generally exceeded 105copies/ml among patients with HBeAg positive chronic hepatitis and may be as high as 1010copies/ml [1], [2], [4], [5], [6], [7], [8], but the correlation between serum HBV DNA levels and histologic activity was poor [7]. Serum HBV DNA levels tended to be lower (104–108copies/ml) among patients with HBeAg negative chronic hepatitis B (HBeAg negative, anti-HBe positive, elevated ALT) [4], [5] and may at times be undetectable in non-PCR based assays [9], [12]. One study found that HBeAg negative patients with high serum HBV DNA levels (>107copies/ml) were more likely to have increased histologic activity and fibrosis scores but only six patients had high HBV DNA levels in that study [7]. Reports on the relation between precore and core promoter variants and serum HBV DNA levels and liver disease are inconclusive [4], [7], [9], [13]. The inability to demonstrate a correlation between serum HBV DNA levels and liver disease is in part related to the fact that HBV induced liver damage is predominantly immune-mediated. However, many of the earlier studies have also been hampered by the lack of sensitivity and/or standardization of the HBV DNA assays used, and the absence of serial serum samples and/or liver histology.

Recent availability of commercial quantitative PCR assays and establishment of international standards for HBV DNA assays have enabled some of the questions regarding HBV DNA levels and liver disease to be re-examined. In this issue of the Journal, Martinot-Peignoux et al. [14] determined serum HBV DNA levels of inactive HBsAg carriers (HBeAg negative, anti-HBe positive, with persistently normal ALT levels) using a commercially available PCR assay (Cobas Amplicor HBV Monitor™), which has a detection limit of 200copies/ml. They found that 71 (84%) of 85 inactive carriers had detectable HBV DNA, but only two (2%) had levels >105copies/ml. Annual testing of 38 patients followed for 1–6 years found that serum HBV DNA levels remained stable in most patients, with median values ranging from 103 to 104copies/ml. Only one (3%) patient had HBV DNA level >105copies/ml. The authors concluded that their results support the NIH recommendation of 105copies/ml as a cut-off HBV DNA level for differentiating inactive carriers from those with chronic hepatitis B. However, this study did not include patients with chronic hepatitis B. Thus, the reliability of an HBV DNA level of 105copies/ml in differentiating inactive carriers from patients with chronic hepatitis B cannot be established. Indeed, a recent report from Greece found that 13% of 134 patients with HBeAg negative chronic hepatitis had serum HBV DNA levels less than 105copies/ml indicating that a cut-off value of 105copies/ml would lead to misclassification of these patients and possibly denial of treatment [15]. In this study, all 68 inactive carriers but only 11% of patients with HBeAg negative chronic hepatitis B had serum HBV DNA levels <3×104copies/ml. The authors concluded that a cut-off HBV DNA level of 3×104copies/ml is more appropriate for differentiating inactive carriers from patients with HBeAg negative chronic hepatitis B but this study was based on serum HBV DNA levels taken at a single time point. Clearly, more studies on a larger number of patients followed over a longer period of time are needed to validate this cut-off value.

Establishment of a threshold serum HBV DNA level that is associated with liver injury can be important for predicting prognosis and for defining indications for treatment. However, given the variable nature of chronic HBV infection, it is possible that this threshold level may be different for each individual and may vary with time depending on the host immune status and other exogenous factors. Thus, the main objective of defining such a threshold level, if it exists, is to identify individuals with very low risk of progressive liver disease, in whom current treatment offers little or no benefit, and who may require less frequent monitoring. Nevertheless, repeat testing is necessary even for inactive carriers as serum HBV DNA levels may fluctuate with time. In addition, decision on treatment should not be based simply on serum HBV DNA level. Other factors such as histologic activity, likelihood of response and other co-morbid conditions should be considered.

With the recognition that nosocomial transmission of HBV infection can occur from HBeAg negative health care workers, who have chronic HBV infection [16], some countries such as England, have attempted to establish guidelines on restrictions on practice based on serum HBV DNA levels [17]. This policy must be carefully reviewed because of the wide fluctuations in serum HBV DNA levels particularly in patients with HBeAg negative chronic hepatitis B.

A key element in the evaluation of new treatment of chronic hepatitis B is to determine the degree of reduction in serum HBV DNA levels. The availability of quantitative PCR assays for HBV DNA permits more accurate assessment of dose–response effect and comparison of the potencies of various antiviral compounds. In addition, the ability to detect lower HBV DNA levels enables investigators and physicians to determine if continued treatment is associated with further decrease in virus levels as well as earlier detection of relapse or breakthrough infection. Thus, quantitative PCR assays have improved our ability to monitor treatment response, but several important questions have arisen. First, to what level should HBV DNA be reduced to ensure sustained virologic and clinical remission? The answer to this question is predicated on the availability of potent antiviral agents that when used singly or in combination will be able to decrease serum HBV DNA to the desired level in the vast majority of patients. Such level has not been determined. Ideally, one would like to reduce serum HBV DNA to levels undetectable by the most sensitive PCR assays but current treatment rarely achieves this goal. Second, to what level should HBV DNA be reduced to achieve HBeAg seroconversion? To date, only one study had addressed this issue. In a study of 23 patients who received lamivudine therapy, six (50%) of 12 patients whose serum HBV DNA decreased to <104copies/ml developed HBeAg seroconversion versus none of 11 whose serum HBV DNA remained >104copies/ml [18]. This study suggested that there may be a threshold HBV DNA level associated with lamivudine induced HBeAg seroconversion. In an ongoing study of patients with spontaneous or interferon induced HBeAg seroconversion, we found that serum HBV DNA levels ranged from 104 to 108copies/ml at the time when HBeAg first became undetectable (personal observations). Thus, further studies are needed to determine if HBeAg seroconversion is associated with a specific HBV DNA level. Third, how should treatment response of HBeAg negative chronic hepatitis B be defined? Patients with HBeAg negative chronic hepatitis B tend to have lower serum HBV DNA levels that may at times be undetectable using non-PCR based assays, and HBeAg loss or seroconversion cannot be used as an end-point. Clearly, quantitative PCR assays for HBV DNA are very important in monitoring response of these patients, but it is not known if reduction in serum HBV DNA to levels undetectable by PCR assays is necessary or sufficient for sustained remission and if treatment can be withdrawn after serum HBV DNA become undetectable by PCR assay.

In summary, quantitative PCR assays for serum HBV DNA have provided many answers and also raised new questions. Studies similar to that of Martinot-Peignoux et al. should be repeated in more diverse patient populations to address the questions raised so results of these tests can be appropriately interpreted and used in the management of patients with chronic HBV infection.

Back to Article Outline

References 

  1. Loeb KR, Jerome KR, Goddard J, Huang ML, Cent A, Corey L. High-throughput quantitative analysis of hepatitis B virus DNA in serum using the TaqMan fluorogenic detection system. Hepatology. 2000;32:626–629
  2. Noborg U, Gusdal A, Horal P, Lindh M. Levels of viraemia in subjects with serological markers of past or chronic hepatitis B virus infection. Scand J Infect Dis. 2000;32:249–252
  3. Michalak TI, Pasquinelli C, Guilhot S, Chisari FV. Hepatitis B virus persistence after recovery from acute viral hepatitis. J Clin Invest. 1994;93:230–239
  4. Niitsuma H, Ishii M, Miura M, Kobayashi K, Toyota T. Low level hepatitis B viremia detected by polymerase chain reaction accompanies the absence of HBe antigenemia and hepatitis in hepatitis B virus carriers. Am J Gastroenterol. 1997;92:119–123
  5. Gerken G, Gomes J, Lampertico P, Colombo M, Rothaar T, Trippler M, et al.  Clinical evaluation and applications of the Amplicor HBV MonitorTM test, a quantitative HBV DNA PCR assay. J Virol Methods. 1998;74:155–165
  6. Fujiwara K, Yokosuka O, Ehata T, Chuang WL, Imazeki F, Saisho H, et al.  The two different states of hepatitis B virus DNA in asymptomatic carriers: HBeAg-antigen-positive versus anti-HBe-positive asymptomatic carriers. Dig Dis Sci. 1998;43:368–376
  7. Lindh M, Horal P, Dhillon AP, Norkrans G. Hepatitis B virus DNA levels, precore mutations, genotypes and histological activity in chronic hepatitis B. J Viral Hepatitis. 2000;7:258–267
  8. Kessler HH, Preininger S, Stelzl E, Daghofer E, Santner BI, Marth E, et al.  Identification of different states of hepatitis B virus infection with a quantitative PCR assay. Clin Diagn Lab Immun. 2000;7:298–300
  9. Chan HLY, Leung NWY, Hussain M, Wong ML, Lok ASF. Hepatitis B e antigen-negative chronic hepatitis B in Hong Kong. Hepatology. 2000;31:763–768
  10. Lok AS, Heathcote EJ, Hoofnagle JH. Management of Hepatitis B 2000, Summary of a Workshop. Gastroenterology. 2001;120:1828–1853
  11. Chu CM, Karayiannis P, Fowler MJ, Monjardino J, Liaw YF, Thomas HC. Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum. Hepatology. 1985;5:431–434
  12. Jardi R, Buti M, Rodriguez-Frias F, Cortina M, Esteban R, Guardia J, et al.  The value of quantitative detection of HBV-DNA amplified by PCR in the study of hepatitis B infection. J Hepatol. 1996;24:680–685
  13. Agalar C, Diri C, Usubutun S, Agalar F, Turkyilmaz R. The role of HBV DNA and liver histopathology in HBsAg carriers. Hepatogastroenterology. 1997;44:1196–1199
  14. Martinot-Peignoux M, Boyer N, Colombat M, Akremi R, Pham B-N, Castelnau C, et al.  Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers. J Hepatol. 2002;36:543–546
  15. Manesis EK, Papatheodoridis GV, Hadziyannis SJ. Serum HBV DNA levels in inactive hepatitis B virus carriers. Gastroenterolgy. 2002; (in press)
  16. The Incident Investigation Team and Others . Transmission of hepatitis B to patients from four infected surgeons without hepatitis B e antigen. N Engl J Med. 1997;336:178–184
  17. Hepatitis B infected health care workers. Department of Health: Health Service Circular: HSC 2000/020; 23 June 2000.
  18. Gauthier J, Bourne EJ, Lutz MW, Crowther LM, Dienstag JL, Brown NA, et al.  Quantitation of hepatitis B viremia and emergence of YMDD variants in patients with chronic hepatitis B treated with lamivudine. J Infect Dis. 1999;180:1757–1762

PII: S0168-8278(02)00039-9

doi:10.1016/S0168-8278(02)00039-9

Journal of Hepatology
Volume 36, Issue 4 , Pages 549-551, April 2002

Article Tools