An estimated 350 million individuals worldwide and 1.25 million in the United States are infected with hepatitis B virus (HBV).1,2 Patients with chronic HBV infection have a protean presentation including asymptomatic “carrier” status, chronic hepatitis, cirrhosis, hepatocellular cancer (HCC), and very rarely, fulminant liver failure. Worldwide, the most common mode of infection is perinatal transmission (vertical transmission), but in developed countries like the United States and many European countries, the infection occurs largely during adulthood.
Chronic HBV infection may present clinically in 4 different ways: (1) immune tolerant phase, which typically occurs after perinatal transmission, characterized by the presence of HBeAg, high serum levels of HBV DNA but normal alanine aminotransferases (ALT) and minimal or no inflammation on biopsy; (2) chronic hepatitis B (CHB) phase (immune reactive phase), which may occur after many years of immune tolerant phase in those who acquired the infection perinatally and earlier after infection in those who acquired the infection in adulthood, characterized by raised ALT, elevated HBV DNA, HBeAg, and a histologic evidence of chronic hepatitis without or with varying degrees of fibrosis; (3) inactive HBV carrier phase, where patients have HBsAg, normal ALT, HBV DNA <104 copies/mL, negative HBeAg, positive HBeAb, and inactive disease on liver histology3,4; and (4) HBeAg-negative chronic, often relapsing, hepatitis characterized by mutation at precore or core-promoter region, leading to replication and liver injury in the absence HBeAg and presence of HBeAb. This happens perhaps in only a minority of patients, but over time it has become the dominant type in many countries. It is assumed that all chronic HBeAg-negative hepatitis patients evolve from mutation of HBV strain that produce HBeAg (“wild-type”) to “mutants” that fail to produce HBeAg and yet retain the ability to replicate and cause liver injury by immune-mediated mechanism. It has been suggested that de nova infection in adulthood or perinatal infection rarely leads to chronic hepatitis although it may cause acute liver disease or even liver failure.
During the natural history of HBV infection, HBV DNA levels are highest (>108 copies/mL) in immune tolerant phase and lowest in inactive status. During immune reactive phase, DNA levels start declining in many patients with spontaneous clearance of HBeAg and development of HBeAb in 5% to 10% patients per year. However, those who fail to clear HBeAg during this phase may develop more severe and progressive liver disease resulting in cirrhosis. Once patients becomes inactive carriers (HBeAg-negative/HBeAb-positive nonreplicating state), most (approximately 80%) remain as inactive carriers, but about 10% to 20% may revert back to actively replicating phase accompanied by reverting back to HBeAg-positive status, elevated ALT, and ongoing liver inflammation.5,6 A variable (depending on the genotype) proportion of HBV mutate during its long natural course at precore (G1896A lead to premature stop codon that prevents HBeAg production) or core-promoter (2 nucleotide changes, A1762T and G1764A, affects HBeAg production at transcriptional level) regions leading to HBeAb-positive status but with continued replication without producing HBeAg (referred as HBeAg-negative strains or “precore/core-promoter mutant”). These patients (HBeAg negative/HBeAb positive, replicating state) may clinically remain unrecognized with chronic hepatitis or may present with rapidly worsening liver disease; in these patients, HBV DNA levels may fluctuate and may be lower than those HBeAg-positive patients with hepatitis.
At the 2000 NIH workshop, a HBV DNA >105 copies/mL (approximately 20,000 IU/L) was arbitrarily chosen to differentiate between inactive carriers and those with chronic hepatitis.3 It is important to note that this was an “arbitrary” cut-off based on the measurements of HBV DNA levels by the hybridization techniques where detection limits were higher (HBV DNA levels of <105 copies/mL) than polymerase chain reaction-based measurements currently used in most countries. When it comes to deciding on the treatment of CHB with active liver injury, the American Association for Study of the Liver (AASLD) guidelines suggest HBV DNA >105 copies/mL as the cut-off for considering the treatment.1 The European Association for the Study of the Liver (EASL) and the Asian Pacific Association for the Study of the Liver (APASL) have issued somewhat similar recommendations.7,8 Despite these recommendations from the 3 associations, 2 important questions remain unanswered. Is there any consistent relationship between HBV DNA levels and histology and as a corollary to that, do HBV DNA levels of 105 copies/mL accurately differentiate between inactive carriers and those with active liver disease (CHB)?
In the February issue of the journal, Nabuco et al9 attempt to answer the above questions. They describe the correlation between HBV DNA levels and liver histology in 47 blood donors from Brazil with detectable viremia (>1000 copies/mL) as assessed by Amplicor HBV Monitor Roche assay. Of these 47, 20 (12 HBeAg positive) had histologic evidence of hepatitis on histology, and these patients had very high median HBV DNA (25,260,000 copies/mL) levels. In contrast, 27 patients (4 HBeAg positive) with normal histology, median HBV DNA levels were 9480 copies/mL. Overall, authors found a significant correlation between HBV DNA levels and hepatic necroinflammation (r=0.59, P<0.001) and fibrosis (r=0.50, P<0.001). Among 8 HBeAg-negative patients with histologic evidence of liver disease, 5 had evidence of liver disease (1 cirrhosis and 4 mild chronic hepatitis) despite HBV DNA <30,000 copies/mL and 4 of these patients had normal ALT on the 3 consecutive measurements at 2-month intervals.
What can we learn about the natural history of HBV from a cross-sectional study of this nature? This study has many major limitations and therefore, it is difficult to draw any firm conclusions. The sample size was very small, and HBV DNA levels were measured only once. It is known that HBV DNA levels may fluctuate, especially in HBeAg-negative patients with precore/core-promoter mutants. Another possibility is that some of these patients (HBeAg positive) may be in the transition stage between immune reactive and inactive phase as discussed earlier. Selection bias could also influence the results unless consecutive patients with HBsAg were recruited.
Other authors have also attempted to assess the correlation between HBV DNA levels and liver histology. Zavaglia et al10 found no correlation between HBV DNA levels and histology in HBeAg-positive patients, but HBeAg-negative patients with piecemeal necrosis had higher HBV DNA levels. Yuen et al11 also reported similar results in their Asian cohort with HBV. Furthermore, they also observed that although the majority of HBeAg-negative patients with HBV DNA levels <10×5 had mild necroinflammation with no fibrosis, 14.3% had established fibrosis, and those with mutation at core-promoter region had more severe histologic changes. Similar observations have also been made in Northern European patients where more severe necroinflammation was found in patients with genotype C compared with genotypes A and D.12 In all these 3 studies, elevated ALT correlated with liver inflammation.10–12 Nabuco et al9 did not attempt to correlate ALT with necroinflammation in the liver biopsy. A correlation between HBV DNA levels and liver histology has been previously reported in HBeAg-negative Asian and Mediterranean subjects. However, it is not known whether this is applicable to other ethnic groups because of the differences in the prevalence of HBV genotypes and modes of transmission. The consistent lack of correlation between HBV DNA levels and liver histology in HBeAg-positive subjects observed by many investigators could be because many of these studied subjects could have been in the immune tolerance phase where there may be high viral replication with no/minimal immune response to it.11
An important question that remains unanswered is whether the cut-offs currently in practice (HBV DNA levels of 10×5 copies/mL) accurately differentiate between inactive HBV carriers and those with CHB? Data by Martinot-Peignoux et al13 supported this cut-off level. In their 85 consecutive HBsAg-positive/HBeAg-negative patients with persistently normal ALT, the median HBV DNA was 1300 copies/mL and 98% had HBV DNA <100,000 copies/mL, and histologic lesions were mild in all cases. In this study, no correlation was seen between histology and HBV DNA levels, though all patients had normal ALT. However, some recent data challenge this arbitrary cut-off value of 105 copies/mL to differentiate between inactive carriers and those with CHB. In a study of 196 patients with HBeAg-negative chronic HBV infection (62 inactive carriers, 134 with CHB), serum ALT levels were normal at baseline in 25 of 134 (18.7%) patients with CHB patients, whereas it remained normal throughout the follow-up in all inactive carriers.14 HBV DNA <30,000 copies/mL was observed in 14 (10.5%) and <100,000 copies/mL in 17 (12.9%) HBeAg-negative CHB patients, whereas it was <30,000 copies/mL in all inactive carriers (undetectable in 14). In particular, HBV DNA levels were <100,000 copies/mL in 8 (32%) and <30,000 copies/mL in 5 (20%) of the 25 patients with HBeAg-negative CHB with normal baseline ALT values. On the basis of these observations, authors suggested that the cut-off DNA levels to differentiate between CHB and inactive carriers should be set at 30,000 copies/mL. However, the absence of liver histology was a major limitation of this study. More recently, Chu et al15 showed that 45% of their patients who were HBeAg negative had elevated ALT but HBV DNA <105. Finally, Yuen et al11 and Nabuco et al9 observed fibrosis in 14.3% and 25% of their cohort, respectively, with HBeAg-negative CHB who had HBV DNA levels <10×5 copies/mL. Despite the limitations of all the above studies, it seems that the cut-off currently recommended (HBV DNA level of 100,000 copies/mL) may not have adequate specificity and accuracy to differentiate between inactive carriers and those with CHB.
Finally, what is the clinical relevance or consequence of identifying a relationship between HBV DNA levels and histology? This is difficult to predict in the absence of prospective studies with serial biopsies. However, in a recent population-based prospective cohort study of 3582 untreated HBV patients from Taiwan, during a mean follow-up of 11 years, the cumulative incidence of cirrhosis increased with HBV DNA levels and ranged from 4.5% to 36.2% with a HBV DNA load of <300 copies/mL and >106 copies/mL, respectively.16 This increased risk was independent of HBeAg status and serum ALT level. In addition, Chen et al17 reported that the elevated HBV DNA levels (>10,000 copies/mL) to be a strong predictor of HCC independent of HBeAg status, serum ALT levels, and liver cirrhosis. Of the 164 cases of HCC observed in this study (mean follow-up 11.4 y), 48 occurred (38%) in those with HBV DNA levels <100,000 copies/mL.
Although failure to clear HBeAg is associated with an increased risk to develop cirrhosis and HCC, loss of HBeAg may not always have a benign course as many of these patients may have precore/core-promoter mutants that may be associated with fluctuating HBV DNA levels and more rapid progression of liver disease. Longitudinal follow-up and better assessment of HBV status is critical to address prognosis in these patients. From a practical point of view, clinicians who take care of patients should understand that the presence of HBeAg negative/HBeAb-positive status does not imply nonreplicating status. Presence of high HBV DNA levels may suggest that these patients may have a HBV strain that has mutated at precore/core-promoter region. A provocative study suggested that even the loss of HBsAg might not always guarantee a good prognosis.18,19 In this study, Huo et al18 found the presence of HBV DNA in 31.4% of chronic carriers (n=55) after they had spontaneously cleared the HBsAg, and during a mean follow-up of 23 months after HBsAg clearance, 32% developed a liver related complication (11 HCC, 6 cirrhosis, and 1 subfulminant hepatic failure). The limitations of this study were that the HBV DNA was assessed too soon after HBsAg clearance (<2 mo) and about 50% had presence of other hepatotrophic viruses. It is difficult to explain complications other than HCC after clearance of HBsAg in the absence of other insults to the liver. It is therefore important that future studies that attempt to correlate HBV DNA levels, HBeAg status, ALT levels, and genotypes attempt to adjust for other confounding risk factors (nonalcoholic fatty liver disease, alcohol, etc) that may also cause progressive liver disease.
In conclusion, for most of those who are HBeAg positive, no correlation will be observed between HBV DNA levels and histology, especially if they are in the immune tolerant phase. For those whose are HBeAg negative, there may indeed be a correlation, though this needs to be confirmed by larger prospective studies. Overall, irrespective of HBeAg status, there seems to be a consistent correlation between ALT and liver inflammation. The issue whether a HBV DNA of >105 copies/mL accurately differentiate between inactive carrier and those with CHB remains contentious because of paucity of good data. From a practical point of view, for most of the patients, this will probably be an acceptable cut-off. However, there will be few CHB patients who will have an active histology despite having HBV DNA <105 copies/mL, and a minority of these patients may have normal ALT, and some of these patients may progress indolently to cirrhosis. Longitudinal follow-up is critical to understand the status of the disease and prognosis, and controversy in this area suggests that patients with chronic HBV should be followed carefully for signs of chronic liver disease irrespective of HBeAg status.
To address some of the unanswered questions, it is important to prospectively recruit patients with CHB who are HBeAg negative and have HBV DNA <105copies/mL, and perform serial biopsies in all patients with elevated ALT. Only then, we can assess the natural history and outcomes of the disease in this subgroup. Until such data are available, it may not be unreasonable to accept an HBV DNA of 105 copies/mL as the cut-off to differentiate between those who are inactive carriers versus those who have CHB. In doubtful cases, one should have a low threshold for performing a liver biopsy. However, irrespective of HBV DNA and ALT levels, it is of paramount importance that all patients who are HBsAg positive be closely followed-up with regular blood tests and liver imaging for early.
1. Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations. AASLD Practice Guideline. Hepatology. 2004;39:857–861.
2. McQuillan GM, Townsend TR, Fields HA, et al. Seroepidemiology of hepatitis B virus infection in the United States 1976 to 1980. Am J Med. 1989;87:5S–10S.
3. Lok AS, Heathcote EJ, Hoofnagle JH. Management of hepatitis B: 2000, summary of a workshop. Gastroenterology. 2001;120:1828–1853.
4. McMahon BJ. The natural history of chronic hepatitis B virus infection. Sem Liv Dis. 2004;24(suppl 1):17–21.
5. Hadziyannis SJ, Vassilopoulos D. Immunopathogenesis of hepatitis B e antigen chronic hepatitis B infection. Antiviral Res. 2001;52:91–98.
6. Brunetto MR, Oliveri F, Rocca G, et al. Natural course and response to interferon of chronic hepatitis B accompanied by antibody to hepatitis B e antigen. Hepatololgy. 1989;10:198–202.
7. The EASL Jury. EASL International Consensus Conference on hepatitis B 13-14 September, 2002, Geneva, Switzerland. Consensus statement (short version). J Hepatol. 2003;38:533–540.
8. Liaw YF, Leung N, Guan R, et al. Asian-Pacific consensus update working party on chronic hepatitis B. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2005 update. Liver Int. 2005;25:472–489.
9. Nabuco LC, Villela-Nogueira CA, Perez RM, et al. HBV DNA levels in HBsAg positive blood donors and its relationship with liver histology. J Clin Gastroenterol. 2007;41:194–198.
10. Zavaglia C, Mondazzi L, Maggi G, et al. Are alanine aminotransferase, hepatitis B virus DNA or IgM antibody to hepatitis B core antigen serum levels predictors of histological grading in chronic hepatitis B? Liver. 1997;17:83–87.
11. Yuen MF, Ng IO, Fan ST, et al. Significance of HBV DNA levels in liver histology of HBeAg and Anti-HBe positive patients with chronic hepatitis B. Am J Gastroenterol. 2004;99:2032–2037.
12. Lindh M, Horal P, Dhillon AP, et al. Hepatitis B virus DNA levels, precore mutations, genotypes and histological activity in chronic hepatitis B. J Viral Hepat. 2000;7:258–267.
13. Martinot-Peignoux M, Boyer N, Colombat M, et al. Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers. J Hepatol. 2002;36:543–546.
14. Manesis EK, Papatheodoridis GV, Sevastianos V, et al. Significance of hepatitis B viremia levels determined by a quantitative polymerase chain reaction assay in patients with hepatitis B e antigen-negative chronic hepatitis B virus infection. Am J Gastroenterol. 2003;98:2261–2267.
15. Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. Hepatology. 2002;36:1408–1415.
16. Iloeje UH, Yang HI, Su J, et al., Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-In HBV (the REVEAL-HBV) Study Group. Predicting cirrhosis risk based on the level of circulating hepatitis B viral load. Gastroenterology. 2006;130:678–686.
17. Chen CJ, Yang HI, Su J, et al., REVEAL-HBV Study Group. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA. 2006;295:65–73.
18. Huo TI, Wu JC, Lee PC, et al. Sero-clearance of hepatitis B surface antigen in chronic carriers does not necessarily imply a good prognosis. Hepatology. 1998;28:231–236.
19. McMahon BJ. Chronic carriers of hepatitis B virus who clear hepatitis B surface antigen: are they really “off the hook”. Hepatology. 1998;28:265–267.