Clinica Chimica Acta 438 (2015) 266–268
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case Report
Occult hepatitis B virus infection with positive hepatitis B e antigen Zhenge Han a,b,1, Yong Liu b,d,1, Jinshun Pan b,d, Yongchun Bi b, Jingli Liu b,d, Yi-Hua Zhou b,c,d,⁎ a
Department of Laboratory Medicine, East China Sanatorium, Wuxi 214065, Jiangsu Province, China Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China c Department of Infectious Diseases, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China d Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, Nanjing 210008, China b
a r t i c l e
i n f o
Article history: Received 14 July 2014 Received in revised form 3 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Occult HBV infection HBeAg HBsAg mutants
a b s t r a c t Background: Hepatitis B e antigen (HBeAg) is a marker to indicate active replication of hepatitis B virus (HBV). Occult HBV infection (OBI), referred to persistence of HBV DNA in serum and/or liver without detectable serum hepatitis B surface (HBsAg), usually has low HBV DNA levels. The presence of HBeAg in OBI is unusual. Methods: We report 2 patients who presented negative for HBsAg but positive for HBeAg and HBV DNA. HBV markers were quantified in the longitudinal sera in a period of 1–2 years. The HBV DNA sequences were analyzed in 2 patients' sera and 1 patient's liver. Results: Both patients were also positive for total anti-HBs and anti-HBc but negative for anti-HBe and anti-HBc IgM. HBV DNA levels were 234–567 IU/ml in case 1 and 42–1130 IU/ml in case 2. The alignment analysis of the S gene showed that HBV in both patients was genotype C, serotype adr. Cloning analysis of the a determinant of HBsAg showed that the immune escape mutants were predominant in both patients over the follow-up period. The HBV had double mutations (A1762T and G1764A) in the basal core promoter but had no mutation in the pre C/C gene in both patients. Conclusions: The patients with negative HBsAg but positive HBeAg may represent a unique type of OBI. Test for HBeAg would be critical to identifying such type of OBI. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Infection with hepatitis B virus (HBV) is usually diagnosed by the presence of the hepatitis B surface (HBsAg), typically coexistent with antibody against hepatitis B core antigen (anti-HBc), and hepatitis B e antigen (HBeAg) or antibody against HBeAg (anti-HBe). However, occult HBV infection (OBI), defined as the persistence of HBV DNA in serum and/or liver without detectable serum HBsAg, regardless of the status of anti-HBs and anti-HBc, has been widely recognized in various patients and even in healthy persons and accepted blood donors [1]. Based on the Statements from the Taormina expert meeting [1], OBI is characterized with low replication of HBV in liver, usually with HBV DNA b 200 IU/ml in serum, which is the result of HBsAg loss after acute or chronic infections. The Statements also refer to the “false” OBI, which is caused by infection with the S gene escape mutant, with HBV DNA levels comparable to overt infection [1].
⁎ Corresponding author at: Departments of Laboratory Medicine and Infectious Diseases, Nanjing Drum Tower Hospital, 321 Zhong Shan Road, Nanjing 210008, China. Tel.: +86 25 8330 4616x10373; fax: +86 25 8310 5987. E-mail address: [email protected] (Y.-H. Zhou). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cca.2014.09.007 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Previously, we reported an HBsAg negative yet HBeAg positive chronic HBV carrier infected with both wild and mutant viruses; the HBV DNA levels were 370–491 copies/ml [2]. Subsequently, 2 studies reported antiviral-naïve HBV-infected patients with negative HBsAg and positive HBeAg [3,4]; 1 patient was infected with HBV S gene escape mutation G145R, with serum HBV DNA 34–39 IU/ml [3], whereas another patient was infected with the wild virus with serum HBV DNA 103–106 copies/ml [4]. Here we describe two additional antiviralnaïve HBV infected patients who were negative for HBsAg but were still HBeAg positive. Therefore, OBI with positive HBeAg would represent a unique serological profile of HBV infection.
2. Case report Case 1, 56 y-old man, had been diagnosed with chronic hepatitis B and assumed to have resolved the infection because of the persistent normal levels of alanine and aspartate aminiotransferases (ALT and AST), absence of HBsAg and presence of anti-HBs and anti-HBc, detected by the commercial qualitative enzyme immunoassay (EIA) kits (Kehua Biotech), in the routine follow-up in East China Sanatorium. He had not been vaccinated against hepatitis B, and never been treated with interferon or anti-HBV nucleoside/nucleotide analogs. In June 2008,
Z. Han et al. / Clinica Chimica Acta 438 (2015) 266–268
liver function tests showed normal ALT (33 U/l) and AST (25 U/l), and normal total bilirubin level (10.4 μmol/l); HBV serologic markers, detected with chemiluminescent microparticle immunoassay (CMIA, Architect System, Abbott Laboratories), were HBsAg negative, but positive for anti-HBs, anti-HBc, and HBeAg (Table 1). Serum HBV DNA level, detected by a real-time PCR kit (Shenyou Biotech) with 7500 Real-Time PCR System (Applied Biosystems), was 234 IU/ml. In the 2 follow-ups in August 2009 and November 2010 respectively, the ALT and AST and total bilirubin levels were generally normal and the HBV serological pattern remained unchanged, and the serum HBV DNA level was 567 IU/ml (Table 1). Direct sequencing of the S gene amplified by PCR from the serum collected in June 2008 showed that HBV was genotype C and serotype adr (122 K and 160R), and the wild and mutant viruses co-existed in the patient. Further sequencing analysis after the PCR products were inserted into the T-A cloning vectors (Sangon Biotech) showed that there were at least 4 sequences in the a determinant, including one wild type and three mutant sequences (Fig. 1a). Sequencing of the PCR products covering the basal core promoter (BCP) and pre C/C gene from the same serum displayed double mutations (A1762T and G1764A) in BCP, and no mutation in the pre C/C gene. In the followup of November 2010, direct sequencing and T-A cloning of the S gene showed that the a determinant sequences were all mutant type (Fig. 1a). BCP double mutations remained constant and the pre C/C gene did not undergo mutation. Case 2, an 81-y-old man, was hospitalized in Nanjing Drum Tower Hospital January 2010 because he was diagnosed with hepatocellular carcinoma (HCC). In 2007, he was defined with liver cirrhosis and was first found to be positive for anti-HBs, anti-HBc and HBeAg but negative for HBsAg and anti-HBe (all detected by qualitative ELISA) in a local hospital; his status of HBV infection before 2007 was unknown. He had no history of vaccination against hepatitis B or treatment with anti-HBV agents. Anti-HCV was negative both in the local hospital in 2007 and in our hospital. He had no history of alcohol overconsumption. The HBV markers were HBsAg negative, but positive for HBeAg, antiHBs, and anti-HBc, and HBV DNA was 1130 IU/ml (Table 1). Direct sequencing of the PCR products showed that the virus was also genotype C and serotype adr, and there were three residue substitutions in the a determinant (Ile126The, The131Asn and Met133Thr) (Fig. 1b) and double mutations (A1762T and G1764A) in BCP, but no mutations in the pre C/C gene. He underwent hepatectomy on January 19, 2010; pathological examinations revealed that tumor was 3 × 2 × 2 cm in size, and a moderately differentiated hepatocellular carcinoma. The S and pre C/C gene sequences, amplified from liver tissue DNA, were identical to those in the serum (Fig. 1b). During a whole year follow-up, his liver functions were generally normal, the serological pattern remained unchanged, and HBV DNA fluctuated from 42 to 135 IU/ml (Table 1). Cloning analysis of the PCR products in the follow-up showed that the variants harboring the mutations in the a determinant was still predominant (Fig. 1b).
267
3. Discussion The patients we report here were HBsAg negative but positive for HBeAg and HBV DNA; the diagnosis of OBI can be no doubt established. The negativity of HBsAg in the two patients might be associated with the low antigen synthesis and mutations in the a determinant of HBsAg. Based on the Taormina Statements [1], infection with HBV harboring S gene escape mutants, in which HBV DNA levels are usually comparable to those in the overt HBV infection, is defined “false” OBI. Indeed, HBV DNA levels in some OBI patients infected with S escape mutants were almost as high as 105 IU/ml [5]. However, it is not always the case. The two patients reported here generally had low serum HBV DNA levels, although the levels in the one case were sometimes over 103 IU/ml (Table 1). Meanwhile, both cases had S gene escape mutants (Fig. 1a). Previously, we reported an HBsAg negative yet HBeAg positive chronic HBV carrier in whom the wild and mutant viruses co-existed; the HBV DNA levels were 370–491 copies/ml [2], which is approximately equal to 74–98 IU/ml. Paparella et al. also described one patient infected by HBV containing S gene G145R mutation, with serum HBV DNA 34–39 IU/ml [3]. Thus, OBI with S gene escape mutants does not necessarily indicate that the HBV DNA level is comparable to that in the overt infection. In other words, infection with the S gene escape mutants may also be the true OBI. It is well documented that HBeAg is associated with active replication of HBV; however, the serum HBV DNA levels in these two patients were generally low in the follow-up (Table 1). This resembles with the recently documented observations in anti-viral naïve patients with HBeAg positive OBI [2,3]. As the mutations in the S gene overlap in the domain of reverse transcriptase (RT), we translated the nucleotide to amino acid sequences to compare with the wild sequence; case 1 and case 2 had four and six amino acid residues changes respectively, sparsely distributed in the RT domain. However, none of the amino acid changes occurred in the YMDD motif and the N-terminal parts of the palm and finger domains, which are critical for RT activity. Thus, it is less likely that the mutations impaired polymerase functions. On the other hand, the host immune responses may be responsible for the low serum HBV DNA levels. Both patients were anti-HBs positive, which might bind to the HBsAg as well as the virions in the circulation. Chimpanzee experiments demonstrate that anti-HBs against wild HBsAg still have neutralizing activity to the mutant virus [6], although the neutralizing effect may be incomplete [7]. Additionally, T-cell mediated immune response may also play roles in reducing HBV replications since the mutations in BCP were presented in both patients [8]. It is of concern whether the escape mutant viruses in these two patients were caused by immune selection in the natural course or by de novo infection with HBV isolates carrying the mutations in the a determinant of S gene. Since both patients were positive for anti-HBs and anti-HBc, one may assume that the patients were infected with escape mutant virus after they had recovered from a wild-type virus infection. However, the results that both patients were persistently anti-HBe
Table 1 Biochemical, serological and virological follow-up of the two patients with occult hepatitis B infection. Reference
ALT (U/l) AST (U/l) HBsAg (IU/ml) Anti-HBs (mIU/ml) HBeAg (S/Co) Anti-HBe (S/Co) Anti-HBc (S/Co) Anti-HBc IgM HBV DNA (IU/ml)
b40 b40 0.00–0.05 b10 b1.0 N1.0 b1.0 – 0
Case 1
Case 2
June 2008
August 2009
Nov. 2010
Jan. 2010
June 2010
Jan. 2011
33 25 − (0.03) + (48.5) + (254.6) − (9.32) + (12.2) – 234
48 26 − (0.01) + (135.8) + (175.3) − (11.4) + (14.3) Not done Not done
34 24 − (0.01) + (142.4) + (167.3) − (12.4) + (13.5) – 567
12 23 − (0.02) + (67.3) + (12.6) − (2.3) + (12.9) – 1130
14 25 − (0.01) + (45.0) + (9.4) − (1.82) + (11.7) – 42
13 25 − (0.01) + (27.9) + (1.1) − (1.49) + (11.3) – 135
268
Z. Han et al. / Clinica Chimica Acta 438 (2015) 266–268
a
negative in the follow-up do not support this assumption since resolved HBV infection also manifests anti-HBe positive in most cases. Furthermore, case 1 had a known history of HBV infection, and underwent an evolution from the co-existence of wild and mutant sequences to the sole escape mutant in the a determinant during the follow-up period (Fig. 1a). Case 2 had liver cirrhosis and HCC, which were closely associated with HBV infection since he had no other risk factors. Thus, the escape mutants were very likely to be the results of immune selection in the natural course of HBV infection. Identification of OBI patients is important in several clinical aspects such as reactivation in immunosuppressive patients and cause of cryptic chronic liver disease, and particularly in HBV transmission since HBV is still the most frequent transfusion transmitted agent [9]. Together with the previous cases [2–5,10,11], the patients with negative HBsAg but positive HBeAg may represent a unique type of OBI. Test for HBeAg would be critical to identifying such type of OBI. Acknowledgments This study was supported by a grant (QRX11024) from the Department of Health of Nanjing City, Jiangsu Province, China. References
b
Fig. 1. Interpreted amino acid sequences of the a determinant of hepatitis B surface antigen (HBsAg) from the two patients. PCR denotes the direct sequencing of the PCR products. Dashes indicate residues identical to top ones; the ‘2’ indicates the coexistence of two residues at the position. The Arabic digits that followed the sequences represent the clone numbers. (a) Sequence alignment of case 1. The numbers at the top of the lower panel represent the amino acid positions on HBsAg. (b) Sequence alignment of case 2.
[1] Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008;49:652–7. [2] Zhou YH, Zhou J, Li L, et al. A novel hepatitis B virus mutant coexisting with wild type virus in a carrier with negative HBsAg yet positive HBeAg and anti-HBs. J Clin Virol 2009;46:363–6. [3] Paparella C, De Rosa F, Longo R, et al. Appearance of HBeAg in an occult persistent hepatitis B virus infection. Intervirology 2010;53:173–5. [4] Zhang ZH, Li L, Zhao XP, et al. Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance. J Viral Hepat 2011;18:424–33. [5] Huang X, Qin Y, Li W, et al. Molecular analysis of the hepatitis B virus presurface and surface gene in patients from eastern China with occult hepatitis B. J Med Virol 2013; 85:979–86. [6] Ogata N, Cote PJ, Zanetti AR, et al. Licensed recombinant hepatitis B vaccines protect chimpanzees against infection with the prototype surface gene mutant of hepatitis B virus. Hepatology 1999;30:779–86. [7] Kamili S, Sozzi V, Thompson G, et al. Efficacy of hepatitis B vaccine against antiviral drug-resistant hepatitis B virus mutants in the chimpanzee model. Hepatology 2009;49:1483–91. [8] Cento V, Mirabelli C, Dimonte S, et al. Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution. J Gen Virol 2013;94:143–9. [9] Calderón GM, González-Velázquez F, González-Bonilla CR, et al. Prevalence and risk factors of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus in multiply transfused recipients in Mexico. Transfusion 2009;49:2200–7. [10] Feeney SA, McCaughey C, Watt AP, et al. Reactivation of occult hepatitis B virus infection following cytotoxic lymphoma therapy in an anti-HBc negative patient. J Med Virol 2013;85:597–601. [11] Hou J, Wang Z, Cheng J, et al. Prevalence of naturally occurring surface gene variants of hepatitis B virus in nonimmunized surface antigen-negative Chinese carriers. Hepatology 2001;34:1027–34.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case Report
Occult hepatitis B virus infection with positive hepatitis B e antigen Zhenge Han a,b,1, Yong Liu b,d,1, Jinshun Pan b,d, Yongchun Bi b, Jingli Liu b,d, Yi-Hua Zhou b,c,d,⁎ a
Department of Laboratory Medicine, East China Sanatorium, Wuxi 214065, Jiangsu Province, China Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China c Department of Infectious Diseases, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China d Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, Nanjing 210008, China b
a r t i c l e
i n f o
Article history: Received 14 July 2014 Received in revised form 3 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Occult HBV infection HBeAg HBsAg mutants
a b s t r a c t Background: Hepatitis B e antigen (HBeAg) is a marker to indicate active replication of hepatitis B virus (HBV). Occult HBV infection (OBI), referred to persistence of HBV DNA in serum and/or liver without detectable serum hepatitis B surface (HBsAg), usually has low HBV DNA levels. The presence of HBeAg in OBI is unusual. Methods: We report 2 patients who presented negative for HBsAg but positive for HBeAg and HBV DNA. HBV markers were quantified in the longitudinal sera in a period of 1–2 years. The HBV DNA sequences were analyzed in 2 patients' sera and 1 patient's liver. Results: Both patients were also positive for total anti-HBs and anti-HBc but negative for anti-HBe and anti-HBc IgM. HBV DNA levels were 234–567 IU/ml in case 1 and 42–1130 IU/ml in case 2. The alignment analysis of the S gene showed that HBV in both patients was genotype C, serotype adr. Cloning analysis of the a determinant of HBsAg showed that the immune escape mutants were predominant in both patients over the follow-up period. The HBV had double mutations (A1762T and G1764A) in the basal core promoter but had no mutation in the pre C/C gene in both patients. Conclusions: The patients with negative HBsAg but positive HBeAg may represent a unique type of OBI. Test for HBeAg would be critical to identifying such type of OBI. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Infection with hepatitis B virus (HBV) is usually diagnosed by the presence of the hepatitis B surface (HBsAg), typically coexistent with antibody against hepatitis B core antigen (anti-HBc), and hepatitis B e antigen (HBeAg) or antibody against HBeAg (anti-HBe). However, occult HBV infection (OBI), defined as the persistence of HBV DNA in serum and/or liver without detectable serum HBsAg, regardless of the status of anti-HBs and anti-HBc, has been widely recognized in various patients and even in healthy persons and accepted blood donors [1]. Based on the Statements from the Taormina expert meeting [1], OBI is characterized with low replication of HBV in liver, usually with HBV DNA b 200 IU/ml in serum, which is the result of HBsAg loss after acute or chronic infections. The Statements also refer to the “false” OBI, which is caused by infection with the S gene escape mutant, with HBV DNA levels comparable to overt infection [1].
⁎ Corresponding author at: Departments of Laboratory Medicine and Infectious Diseases, Nanjing Drum Tower Hospital, 321 Zhong Shan Road, Nanjing 210008, China. Tel.: +86 25 8330 4616x10373; fax: +86 25 8310 5987. E-mail address: [email protected] (Y.-H. Zhou). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cca.2014.09.007 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Previously, we reported an HBsAg negative yet HBeAg positive chronic HBV carrier infected with both wild and mutant viruses; the HBV DNA levels were 370–491 copies/ml [2]. Subsequently, 2 studies reported antiviral-naïve HBV-infected patients with negative HBsAg and positive HBeAg [3,4]; 1 patient was infected with HBV S gene escape mutation G145R, with serum HBV DNA 34–39 IU/ml [3], whereas another patient was infected with the wild virus with serum HBV DNA 103–106 copies/ml [4]. Here we describe two additional antiviralnaïve HBV infected patients who were negative for HBsAg but were still HBeAg positive. Therefore, OBI with positive HBeAg would represent a unique serological profile of HBV infection.
2. Case report Case 1, 56 y-old man, had been diagnosed with chronic hepatitis B and assumed to have resolved the infection because of the persistent normal levels of alanine and aspartate aminiotransferases (ALT and AST), absence of HBsAg and presence of anti-HBs and anti-HBc, detected by the commercial qualitative enzyme immunoassay (EIA) kits (Kehua Biotech), in the routine follow-up in East China Sanatorium. He had not been vaccinated against hepatitis B, and never been treated with interferon or anti-HBV nucleoside/nucleotide analogs. In June 2008,
Z. Han et al. / Clinica Chimica Acta 438 (2015) 266–268
liver function tests showed normal ALT (33 U/l) and AST (25 U/l), and normal total bilirubin level (10.4 μmol/l); HBV serologic markers, detected with chemiluminescent microparticle immunoassay (CMIA, Architect System, Abbott Laboratories), were HBsAg negative, but positive for anti-HBs, anti-HBc, and HBeAg (Table 1). Serum HBV DNA level, detected by a real-time PCR kit (Shenyou Biotech) with 7500 Real-Time PCR System (Applied Biosystems), was 234 IU/ml. In the 2 follow-ups in August 2009 and November 2010 respectively, the ALT and AST and total bilirubin levels were generally normal and the HBV serological pattern remained unchanged, and the serum HBV DNA level was 567 IU/ml (Table 1). Direct sequencing of the S gene amplified by PCR from the serum collected in June 2008 showed that HBV was genotype C and serotype adr (122 K and 160R), and the wild and mutant viruses co-existed in the patient. Further sequencing analysis after the PCR products were inserted into the T-A cloning vectors (Sangon Biotech) showed that there were at least 4 sequences in the a determinant, including one wild type and three mutant sequences (Fig. 1a). Sequencing of the PCR products covering the basal core promoter (BCP) and pre C/C gene from the same serum displayed double mutations (A1762T and G1764A) in BCP, and no mutation in the pre C/C gene. In the followup of November 2010, direct sequencing and T-A cloning of the S gene showed that the a determinant sequences were all mutant type (Fig. 1a). BCP double mutations remained constant and the pre C/C gene did not undergo mutation. Case 2, an 81-y-old man, was hospitalized in Nanjing Drum Tower Hospital January 2010 because he was diagnosed with hepatocellular carcinoma (HCC). In 2007, he was defined with liver cirrhosis and was first found to be positive for anti-HBs, anti-HBc and HBeAg but negative for HBsAg and anti-HBe (all detected by qualitative ELISA) in a local hospital; his status of HBV infection before 2007 was unknown. He had no history of vaccination against hepatitis B or treatment with anti-HBV agents. Anti-HCV was negative both in the local hospital in 2007 and in our hospital. He had no history of alcohol overconsumption. The HBV markers were HBsAg negative, but positive for HBeAg, antiHBs, and anti-HBc, and HBV DNA was 1130 IU/ml (Table 1). Direct sequencing of the PCR products showed that the virus was also genotype C and serotype adr, and there were three residue substitutions in the a determinant (Ile126The, The131Asn and Met133Thr) (Fig. 1b) and double mutations (A1762T and G1764A) in BCP, but no mutations in the pre C/C gene. He underwent hepatectomy on January 19, 2010; pathological examinations revealed that tumor was 3 × 2 × 2 cm in size, and a moderately differentiated hepatocellular carcinoma. The S and pre C/C gene sequences, amplified from liver tissue DNA, were identical to those in the serum (Fig. 1b). During a whole year follow-up, his liver functions were generally normal, the serological pattern remained unchanged, and HBV DNA fluctuated from 42 to 135 IU/ml (Table 1). Cloning analysis of the PCR products in the follow-up showed that the variants harboring the mutations in the a determinant was still predominant (Fig. 1b).
267
3. Discussion The patients we report here were HBsAg negative but positive for HBeAg and HBV DNA; the diagnosis of OBI can be no doubt established. The negativity of HBsAg in the two patients might be associated with the low antigen synthesis and mutations in the a determinant of HBsAg. Based on the Taormina Statements [1], infection with HBV harboring S gene escape mutants, in which HBV DNA levels are usually comparable to those in the overt HBV infection, is defined “false” OBI. Indeed, HBV DNA levels in some OBI patients infected with S escape mutants were almost as high as 105 IU/ml [5]. However, it is not always the case. The two patients reported here generally had low serum HBV DNA levels, although the levels in the one case were sometimes over 103 IU/ml (Table 1). Meanwhile, both cases had S gene escape mutants (Fig. 1a). Previously, we reported an HBsAg negative yet HBeAg positive chronic HBV carrier in whom the wild and mutant viruses co-existed; the HBV DNA levels were 370–491 copies/ml [2], which is approximately equal to 74–98 IU/ml. Paparella et al. also described one patient infected by HBV containing S gene G145R mutation, with serum HBV DNA 34–39 IU/ml [3]. Thus, OBI with S gene escape mutants does not necessarily indicate that the HBV DNA level is comparable to that in the overt infection. In other words, infection with the S gene escape mutants may also be the true OBI. It is well documented that HBeAg is associated with active replication of HBV; however, the serum HBV DNA levels in these two patients were generally low in the follow-up (Table 1). This resembles with the recently documented observations in anti-viral naïve patients with HBeAg positive OBI [2,3]. As the mutations in the S gene overlap in the domain of reverse transcriptase (RT), we translated the nucleotide to amino acid sequences to compare with the wild sequence; case 1 and case 2 had four and six amino acid residues changes respectively, sparsely distributed in the RT domain. However, none of the amino acid changes occurred in the YMDD motif and the N-terminal parts of the palm and finger domains, which are critical for RT activity. Thus, it is less likely that the mutations impaired polymerase functions. On the other hand, the host immune responses may be responsible for the low serum HBV DNA levels. Both patients were anti-HBs positive, which might bind to the HBsAg as well as the virions in the circulation. Chimpanzee experiments demonstrate that anti-HBs against wild HBsAg still have neutralizing activity to the mutant virus [6], although the neutralizing effect may be incomplete [7]. Additionally, T-cell mediated immune response may also play roles in reducing HBV replications since the mutations in BCP were presented in both patients [8]. It is of concern whether the escape mutant viruses in these two patients were caused by immune selection in the natural course or by de novo infection with HBV isolates carrying the mutations in the a determinant of S gene. Since both patients were positive for anti-HBs and anti-HBc, one may assume that the patients were infected with escape mutant virus after they had recovered from a wild-type virus infection. However, the results that both patients were persistently anti-HBe
Table 1 Biochemical, serological and virological follow-up of the two patients with occult hepatitis B infection. Reference
ALT (U/l) AST (U/l) HBsAg (IU/ml) Anti-HBs (mIU/ml) HBeAg (S/Co) Anti-HBe (S/Co) Anti-HBc (S/Co) Anti-HBc IgM HBV DNA (IU/ml)
b40 b40 0.00–0.05 b10 b1.0 N1.0 b1.0 – 0
Case 1
Case 2
June 2008
August 2009
Nov. 2010
Jan. 2010
June 2010
Jan. 2011
33 25 − (0.03) + (48.5) + (254.6) − (9.32) + (12.2) – 234
48 26 − (0.01) + (135.8) + (175.3) − (11.4) + (14.3) Not done Not done
34 24 − (0.01) + (142.4) + (167.3) − (12.4) + (13.5) – 567
12 23 − (0.02) + (67.3) + (12.6) − (2.3) + (12.9) – 1130
14 25 − (0.01) + (45.0) + (9.4) − (1.82) + (11.7) – 42
13 25 − (0.01) + (27.9) + (1.1) − (1.49) + (11.3) – 135
268
Z. Han et al. / Clinica Chimica Acta 438 (2015) 266–268
a
negative in the follow-up do not support this assumption since resolved HBV infection also manifests anti-HBe positive in most cases. Furthermore, case 1 had a known history of HBV infection, and underwent an evolution from the co-existence of wild and mutant sequences to the sole escape mutant in the a determinant during the follow-up period (Fig. 1a). Case 2 had liver cirrhosis and HCC, which were closely associated with HBV infection since he had no other risk factors. Thus, the escape mutants were very likely to be the results of immune selection in the natural course of HBV infection. Identification of OBI patients is important in several clinical aspects such as reactivation in immunosuppressive patients and cause of cryptic chronic liver disease, and particularly in HBV transmission since HBV is still the most frequent transfusion transmitted agent [9]. Together with the previous cases [2–5,10,11], the patients with negative HBsAg but positive HBeAg may represent a unique type of OBI. Test for HBeAg would be critical to identifying such type of OBI. Acknowledgments This study was supported by a grant (QRX11024) from the Department of Health of Nanjing City, Jiangsu Province, China. References
b
Fig. 1. Interpreted amino acid sequences of the a determinant of hepatitis B surface antigen (HBsAg) from the two patients. PCR denotes the direct sequencing of the PCR products. Dashes indicate residues identical to top ones; the ‘2’ indicates the coexistence of two residues at the position. The Arabic digits that followed the sequences represent the clone numbers. (a) Sequence alignment of case 1. The numbers at the top of the lower panel represent the amino acid positions on HBsAg. (b) Sequence alignment of case 2.
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