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Lamivudine resistant occult HBV: implications for public health?

2003; Elsevier BV; Volume: 38; Issue: 4 Linguagem: Inglês

10.1016/s0168-8278(03)00079-5

1600-0641

Scott Bowden, Angeline Bartholomeusz, Stephen Locarnini,

Hepatitis Viruses Studies and Epidemiology

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]Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely 'occult'?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (491) Google Scholar] and Hu [[2]Hu K.Q. Occult hepatitis B virus infection and its clinical implications.J Viral Hepat. 2002; 9: 243-257Crossref PubMed Scopus (243) Google Scholar]).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 [1Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely 'occult'?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (491) Google Scholar, 2Hu K.Q. Occult hepatitis B virus infection and its clinical implications.J Viral Hepat. 2002; 9: 243-257Crossref PubMed Scopus (243) Google Scholar, 3Conjeevaram H.S. Lok A.S. Occult hepatitis B virus infection: a hidden menace?.Hepatology. 2001; 34: 204-206Crossref PubMed Scopus (135) Google Scholar]. 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]Conjeevaram H.S. Lok A.S. Occult hepatitis B virus infection: a hidden menace?.Hepatology. 2001; 34: 204-206Crossref PubMed Scopus (135) Google Scholar].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]Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely 'occult'?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (491) Google Scholar]. 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]Blackberg J. Kidd-Ljunggren K. Occult hepatitis B virus after acute self-limited infection persisting for 30 years without sequence variation.J Hepatol. 2000; 33: 992-997Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. 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]Michalak T.I. Pardoe I.U. Coffin C.S. Churchill N.D. Freake D.S. Smith P. et al.Occult lifelong persistence of infectious hepadnavirus and residual liver inflammation in woodchucks convalescent from acute viral hepatitis.Hepatology. 1999; 29: 928-938Crossref PubMed Scopus (112) Google Scholar].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]Wallace W.A. Carman W.F. Surface variation of HBV: scientific and medical relevance.Viral Hep Rev. 1997; 3: 5-16Google Scholar]. 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 [7Blum H.E. Galun E. Liang T.J. von Weizsacker F. Wands J.R. Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication.J Virol. 1991; 65: 1836-1842Crossref PubMed Google Scholar, 8Kohno K. Nishizono A. Terao H. Hiraga M. Mifune K. Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter.J Med Virol. 2000; 61: 15-22Crossref PubMed Scopus (23) Google Scholar], 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 [9Liaw Y.-F. Role of hepatitis C virus in dual and triple hepatitis virus infection.Hepatology. 1995; 22: 1101-1108PubMed Google Scholar, 10Sagnelli E. Coppola N. Scolastico C. Filippini P. Santantonio T. Stroffolini T. et al.Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis.Hepatology. 2000; 32: 1106-1110Crossref PubMed Scopus (197) Google Scholar]. The molecular basis for this inhibition is not known. However, Shih et al. [[11]Shih C.M. Lo S.J. Miyamura T. Chen S.Y. Lee Y.H. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells.J Virol. 1993; 67: 5823-5832Crossref PubMed Google Scholar] 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]Cacciola I. Pollicino T. Squadrito G. Cerenzia G. Orlando M.E. Raimondo G. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease.N Engl J Med. 1999; 341: 22-26Crossref PubMed Scopus (581) Google Scholar] others have been unable to find any association between occult infection and the severity of disease in chronic HCV patients [13Fukuda R. Ishimura N. Niigaki M. Hamamoto S. Satoh S. Tanaka S. et al.Serologically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chronic liver disease: clinical and virological significance.J Med Virol. 1999; 58: 201-207Crossref PubMed Scopus (155) Google Scholar, 14Kazemi-Shirazi L. Petermann D. Muller C. Hepatitis B virus DNA in sera and liver tissue of HBsAg negative patients with chronic hepatitis C.J Hepatol. 2000; 33: 785-790Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Kao J.H. Chen P.J. Lai M.Y. Chen D.S. Occult hepatitis B virus infection and clinical outcomes of patients with chronic hepatitis C.J Clin Microbiol. 2002; 40: 4068-4071Crossref PubMed Scopus (138) Google Scholar].In this issue of the Journal, Besisik and colleagues [[16]Besisik F. Karaca Ç. Akyüz F. Horosanlı S. Önel D. Badur S. et al.Occult HBV infection and YMDD variants in hemodialysis patients with chronic HCV infection.J Hepatol. 2003; 38: 506-510Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar] 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]Locarnini S. Hepatitis B virus surface and polymerase gene variants: potential virological and clinical significance.Hepatology. 1998; 27: 294-297Crossref PubMed Scopus (75) Google Scholar].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]Leung N. Treatment of chronic hepatitis B: case selection and duration of therapy.J Gastroenterol Hepatol. 2002; 17: 409-414Crossref PubMed Scopus (73) Google Scholar]. 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]Stuyver L.J. Locarnini S.A. Lok A. Richman D.D. Carman W.F. Dienstag J.L. et al.Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.Hepatology. 2001; 33: 751-757Crossref PubMed Scopus (360) Google Scholar]. 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 [20Melegari M. Scaglioni P. Wands J. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective.Hepatology. 1998; 27: 628-633Crossref PubMed Scopus (339) Google Scholar, 21Ono-Nita S.K. Kato N. Shiratori Y. Masaki T. Lan K.H. Carrilho F.J. et al.YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: a study by in vitro full-length viral DNA transfection.Hepatology. 1999; 29: 939-945Crossref PubMed Scopus (156) Google Scholar]. 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]Torresi J. Earnest-Silveira L. Deliyannis G. Edgtton K. Zhuang H. Locarnini S.A. et al.Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.Virology. 2002; 293: 305-313Crossref PubMed Scopus (187) Google Scholar]. 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]Torresi J. Earnest-Silveira L. Deliyannis G. Edgtton K. Zhuang H. Locarnini S.A. et al.Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.Virology. 2002; 293: 305-313Crossref PubMed Scopus (187) Google Scholar] 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]Kobayashi S. Ide T. Sata M. Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers.J Hepatol. 2001; 34: 584-586Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. Analogous findings of pretreatment resistance mutations have also been reported for HIV infection [[24]Little S.J. Daar E.S. D'Aquila R.T. Keiser P.H. Connick E. Whitcomb J.M. et al.Reduced antiretroviral drug susceptibility among patients with primary HIV infection.J Am Med Assoc. 1999; 282: 1142-1149Crossref Scopus (317) Google Scholar]. 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]Thibault V. Aubron-Olivier C. Agut H. Katlama C. Primary infection with a lamivudine-resistant hepatitis B virus.AIDS. 2002; 16: 131-133Crossref PubMed Scopus (82) Google Scholar]. 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. 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]Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely 'occult'?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (491) Google Scholar] and Hu [[2]Hu K.Q. Occult hepatitis B virus infection and its clinical implications.J Viral Hepat. 2002; 9: 243-257Crossref PubMed Scopus (243) Google Scholar]). 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 [1Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely 'occult'?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (491) Google Scholar, 2Hu K.Q. Occult hepatitis B virus infection and its clinical implications.J Viral Hepat. 2002; 9: 243-257Crossref PubMed Scopus (243) Google Scholar, 3Conjeevaram H.S. Lok A.S. Occult hepatitis B virus infection: a hidden menace?.Hepatology. 2001; 34: 204-206Crossref PubMed Scopus (135) Google Scholar]. 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]Conjeevaram H.S. Lok A.S. Occult hepatitis B virus infection: a hidden menace?.Hepatology. 2001; 34: 204-206Crossref PubMed Scopus (135) Google Scholar]. 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]Brechot C. Thiers V. Kremsdorf D. Nalpas B. Pol S. Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely 'occult'?.Hepatology. 2001; 34: 194-203Crossref PubMed Scopus (491) Google Scholar]. 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]Blackberg J. Kidd-Ljunggren K. Occult hepatitis B virus after acute self-limited infection persisting for 30 years without sequence variation.J Hepatol. 2000; 33: 992-997Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. 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]Michalak T.I. Pardoe I.U. Coffin C.S. Churchill N.D. Freake D.S. Smith P. et al.Occult lifelong persistence of infectious hepadnavirus and residual liver inflammation in woodchucks convalescent from acute viral hepatitis.Hepatology. 1999; 29: 928-938Crossref PubMed Scopus (112) Google Scholar]. 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]Wallace W.A. Carman W.F. Surface variation of HBV: scientific and medical relevance.Viral Hep Rev. 1997; 3: 5-16Google Scholar]. 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 [7Blum H.E. Galun E. Liang T.J. von Weizsacker F. Wands J.R. Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication.J Virol. 1991; 65: 1836-1842Crossref PubMed Google Scholar, 8Kohno K. Nishizono A. Terao H. Hiraga M. Mifune K. Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter.J Med Virol. 2000; 61: 15-22Crossref PubMed Scopus (23) Google Scholar], 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 [9Liaw Y.-F. Role of hepatitis C virus in dual and triple hepatitis virus infection.Hepatology. 1995; 22: 1101-1108PubMed Google Scholar, 10Sagnelli E. Coppola N. Scolastico C. Filippini P. Santantonio T. Stroffolini T. et al.Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis.Hepatology. 2000; 32: 1106-1110Crossref PubMed Scopus (197) Google Scholar]. The molecular basis for this inhibition is not known. However, Shih et al. [[11]Shih C.M. Lo S.J. Miyamura T. Chen S.Y. Lee Y.H. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells.J Virol. 1993; 67: 5823-5832Crossref PubMed Google Scholar] 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]Cacciola I. Pollicino T. Squadrito G. Cerenzia G. Orlando M.E. Raimondo G. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease.N Engl J Med. 1999; 341: 22-26Crossref PubMed Scopus (581) Google Scholar] others have been unable to find any association between occult infection and the severity of disease in chronic HCV patients [13Fukuda R. Ishimura N. Niigaki M. Hamamoto S. Satoh S. Tanaka S. et al.Serologically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chronic liver disease: clinical and virological significance.J Med Virol. 1999; 58: 201-207Crossref PubMed Scopus (155) Google Scholar, 14Kazemi-Shirazi L. Petermann D. Muller C. Hepatitis B virus DNA in sera and liver tissue of HBsAg negative patients with chronic hepatitis C.J Hepatol. 2000; 33: 785-790Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Kao J.H. Chen P.J. Lai M.Y. Chen D.S. Occult hepatitis B virus infection and clinical outcomes of patients with chronic hepatitis C.J Clin Microbiol. 2002; 40: 4068-4071Crossref PubMed Scopus (138) Google Scholar]. In this issue of the Journal, Besisik and colleagues [[16]Besisik F. Karaca Ç. Akyüz F. Horosanlı S. Önel D. Badur S. et al.Occult HBV infection and YMDD variants in hemodialysis patients with chronic HCV infection.J Hepatol. 2003; 38: 506-510Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar] 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]Locarnini S. Hepatitis B virus surface and polymerase gene variants: potential virological and clinical significance.Hepatology. 1998; 27: 294-297Crossref PubMed Scopus (75) Google Scholar]. 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]Leung N. Treatment of chronic hepatitis B: case selection and duration of therapy.J Gastroenterol Hepatol. 2002; 17: 409-414Crossref PubMed Scopus (73) Google Scholar]. 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]Stuyver L.J. Locarnini S.A. Lok A. Richman D.D. Carman W.F. Dienstag J.L. et al.Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.Hepatology. 2001; 33: 751-757Crossref PubMed Scopus (360) Google Scholar]. 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 [20Melegari M. Scaglioni P. Wands J. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective.Hepatology. 1998; 27: 628-633Crossref PubMed Scopus (339) Google Scholar, 21Ono-Nita S.K. Kato N. Shiratori Y. Masaki T. Lan K.H. Carrilho F.J. et al.YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: a study by in vitro full-length viral DNA transfection.Hepatology. 1999; 29: 939-945Crossref PubMed Scopus (156) Google Scholar]. 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]Torresi J. Earnest-Silveira L. Deliyannis G. Edgtton K. Zhuang H. Locarnini S.A. et al.Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.Virology. 2002; 293: 305-313Crossref PubMed Scopus (187) Google Scholar]. 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]Torresi J. Earnest-Silveira L. Deliyannis G. Edgtton K. Zhuang H. Locarnini S.A. et al.Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.Virology. 2002; 293: 305-313Crossref PubMed Scopus (187) Google Scholar] 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]Kobayashi S. Ide T. Sata M. Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers.J Hepatol. 2001; 34: 584-586Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. Analogous findings of pretreatment resistance mutations have also been reported for HIV infection [[24]Little S.J. Daar E.S. D'Aquila R.T. Keiser P.H. Connick E. Whitcomb J.M. et al.Reduced antiretroviral drug susceptibility among patients with primary HIV infection.J Am Med Assoc. 1999; 282: 1142-1149Crossref Scopus (317) Google Scholar]. 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]Thibault V. Aubron-Olivier C. Agut H. Katlama C. Primary infection with a lamivudine-resistant hepatitis B virus.AIDS. 2002; 16: 131-133Crossref PubMed Scopus (82) Google Scholar]. 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.