Journal of Hepatology
Volume 38, Issue 4 , Pages 526-528, April 2003

Lamivudine resistant occult HBV: implications for public health?

Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Vic. 3051, Australia

See Article, pages 506–510

Article Outline

 

Few serological assays have played a more crucial role in public health than the hepatitis B surface antigen (HBsAg) test. It became the primary assay for the diagnosis of current and chronic hepatitis B virus (HBV) infection and its suitability for mass screening markedly improved the safety of blood stocks in the transfusion service. Loss of HBsAg and the appearance of its corresponding antibody, anti-HBs, are characteristic of resolution of HBV infection. Along with hepatitis B e antigen seroconversion and the development of antibody to the HBV core protein (anti-HBc), this is indicative of elimination of virus and is supported by clinical, histological and biochemical improvement. However, as described in several case reports, a small proportion of patients show clinical evidence of ongoing HBV infection despite developing such serological profiles (see reviews by Brechot and colleagues [1] and Hu [2]).

This apparent paradox has been largely resolved by advances in nucleic acid testing, in particular polymerase chain reaction, which has shown that HBV DNA can be detected after apparent recovery from acute hepatitis and even in some patients with no serological markers of HBV exposure [1], [2], [3]. The terminology of occult HBV infection has been applied to patients with detectable HBV DNA and undetectable HBsAg. This definition does have deficiencies in that several HBV DNA detection technologies are available with different levels of sensitivity. Additionally, while samples may have undetectable HBsAg, this may also be a reflection of the specificity and sensitivity of a particular assay. Moreover, the definition of occult HBV infection does not take into consideration the presence or absence of other HBV markers such as anti-HBc and anti-HBs which may be useful in further defining this clinical entity [3].

Several mechanisms have been proposed to explain the persistence of HBV DNA and lack of detectable HBsAg which occurs with occult infection. Perhaps a clue lies in the one characteristic which seems to be consistently found in many of the studies on occult infection, namely a low level of viral replication, generally <103 copies/ml [1]. Is this due to some property of the infecting virus, the host response or a combination of both? Perhaps deficiencies in the host immune response allow a modest level of virus replication, or conversely, a strong and enduring cellular response may require the persistence of limited amounts of virus. In a 30 year follow-up of an outbreak of hepatitis in Southern Sweden, HBV DNA was detected in the liver but not the serum of two patients with serologically verified acute self-limited hepatitis [4]. Furthermore, using the woodchuck hepatitis virus (WHV) as an animal model for HBV, it has been shown that after recovery from acute WHV infection, a lifelong occult infection persisted with low level replication in liver and lymphoid tissue [5].

Investigators have looked for virus mutations in the surface gene which may cause alterations in HBsAg antigenicity and thus, impair its serological detection or its ability to be neutralized by anti-HBs [6]. However, these mutations appear to be uncommon and in the majority of cases of occult infection there are no instances of sequence changes to the important HBsAg epitopes. Besides mutations directly affecting these epitopes, mutations affecting the HBV regulatory elements may also be a possible cause of down regulation of replication. Again, while there are reports of such mutations and even some functional studies to demonstrate reduced replication [7], [8], it is likely that these are also rare.

Finally, there is mounting evidence of occult HBV infection and a low level of viral replication associated with HCV co-infection. HBV/HCV co-infection is common; the viruses can share the same route of transmission and there are geographical regions where both viruses are endemic. Several groups have reported apparent virus interference between HBV and HCV which may result in a lower level of HBV replication [9], [10]. The molecular basis for this inhibition is not known. However, Shih et al. [11] showed a reduced expression of HBV transcripts by HCV core protein in co-transfection studies. The clinical significance of occult HBV infection in chronic HCV infection is a subject of controversy. Although a role has been proposed in exacerbating liver cirrhosis [12] others have been unable to find any association between occult infection and the severity of disease in chronic HCV patients [13], [14], [15].

In this issue of the Journal, Besisik and colleagues [16] have investigated the prevalence and clinical impact of occult HBV infection in hemodialysis patients with chronic HCV co-infection. Twelve of 33 patients were shown to have occult infection and of significance was that in six of the 12 patients, HBV DNA was shown to have mutations associated with lamivudine (LMV) resistance, even though none of the patients had undergone LMV therapy. This study raises several issues regarding the potential transmission of LMV resistant HBV in the hemodialysis setting. In addition, the potential contribution of the LMV resistant mutations to the lack of HBsAg detection and/or low level of HBV replication in occult HBV must also be considered [17].

The advent of nucleoside/nucleotide analogue treatment for chronic HBV infection, such as LMV, has resulted in the expansion of otherwise minor quasispecies with mutations in the HBV polymerase gene. Although a potent inhibitor of HBV replication, LMV monotherapy has been associated with an incidence of resistance of 38 and 67% after 2 and 4 years, respectively [18]. LMV resistance in HBV has been mapped to a mutation which changes the methionine of the YMDD motif in Domain C of the reverse transcriptase (rt) sequence of the polymerase gene at amino acid position rt204 to isoleucine or valine (rtM204I/V) [19]. This mutation also may occur in association with another mutation causing the change rtL180M within Domain B. HBV encoding the LMV resistance mutations rtM204I/V were initially reported to be replication impaired based on in vitro replication yield assays using transient transfection systems [20], [21]. The majority of published assays to assess replication fitness of LMV resistant virus actually measure the yield phenotype of the virus, with limited standardization to eliminate intra- or inter-assay variability. A true replication fitness assay as employed in other virus systems, such as for HIV, is when two or more viruses are introduced into the same cell and there is direct competition whereby the fitter virus becomes the dominant species. Replication fitness assays based on co-infection are yet to be developed for HBV, so the effect of LMV resistance and other mutations on HBV replication during occult infection remain unknown.

Because the reading frames of HBV genes overlap, mutations selected by antiviral therapy in the polymerase gene may result in changes to the major hydrophilic region (MHR) of the envelope gene which could lead to potential anti-HBs escape [22]. The LMV resistance mutations at rtM204V/I code for changes in the envelope gene at sI195M, sW196L, sW196S, or sW196Stop but these are downstream from the MHR. However, the B domain changes induced by LMV resistance do affect this region. Torresi et al. [22] have recently shown a reduction in the antibody binding to a range of S mutants derived from commonly selected LMV-resistant HBV mutants. Expressed proteins containing these mutations had an altered antigenicity and may have the potential to escape neutralization by anti-HBs antibody. In the study by Besisik et al., other changes in the polymerase gene were not reported and therefore any effects on the envelope gene and subsequent effects on HBsAg detection could not be determined.

Hemodialysis is a known risk factor for infection with HBV and HCV. In the study published in this issue of the Journal, it is difficult to determine the source(s) of HBV infection in the hemodialysis patients with chronic HCV co-infection. The patients who appear to have wild-type HBV DNA may have been originally co-infected with HBV and HCV, with viral interference leading to their HBV infection becoming occult. While it is unlikely that the patients with occult infection with LMV resistant HBV developed such changes without the selection pressure exerted by antiviral therapy, similar mutants have been reported in LMV-untreated asymptomatic hepatitis B carriers [23]. Analogous findings of pretreatment resistance mutations have also been reported for HIV infection [24]. On balance, it is more probable that there has been nosocomial spread of LMV resistant virus from other patients on hemodialysis who are been treated for HBV infection. Transmission of LMV resistant HBV has been reported with primary infection of an HIV-HBV co-infected individual [25]. This patient was receiving LMV as a component of highly active antiretroviral therapy at the time of HBV transmission and was not immunized against HBV infection. In the present study, it was not possible to determine the number of potential instances of nosocomial transmission, either with LMV resistant HBV or wild-type virus. Was there one source of LMV resistant virus in which different quasispecies have been selected by the patients, resulting in the three Line Probe assay patterns, or were there three different sources of nosocomial spread? As four of the six patients share the one pattern, the horizontal spread among these patients is a further possibility. These questions could be resolved by sequencing and phylogenetic analysis in conjunction with an epidemiological investigation.

Whether occult HBV infection has any clinical impact in the hemodialysis setting remains to be determined. Nevertheless, from a public health perspective, the study emphasizes some important messages. First, the screening of hemodialysis patients for HBsAg alone appears inadequate for the diagnosis of HBV infection, particularly in patients who are co-infected with HCV. While standardization between sensitive HBV DNA tests remains problematic, their use should be strongly considered as an adjunct to screening. In addition, vaccination of hemodialysis patients who are at risk for HBV infection should be carried out and their anti-HBs status should be determined to minimize the risk of nosocomial spread. Finally, it is not known whether occult HBV should be treated but transmission of HBV from patients with occult infection does occur. The nosocomial spread of LMV resistant virus to uninfected individuals limits the choices as to what the clinician could use if treatment was required.

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PII: S0168-8278(03)00079-5

doi:10.1016/S0168-8278(03)00079-5

Journal of Hepatology
Volume 38, Issue 4 , Pages 526-528, April 2003

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