A Method for the Automated Screening for Hepatitis B Surface Antigen

Abstract. A reverse passive hemagglutination (RPHA-G) technique for the automated screening of hepatitis B surface antigen (HBsAg) on the Groupamatic 360 (Kontron International, Zürich, Switzerland) has been developed. It is more sensitive than manual RPHA (RPHA-M) and nearly as sensitive as radioimmunoassay (RIA); it detects 6 ng/ml of HBsAg, compared to 3 ng/ml by RIA and 25 ng/ml by RPHA-M. Although it is slightly inferior to the other two methods in respect of specificity, the incidence of false-positive reactions was only 0.34%. The test can also be performed manually without the Groupamatic.     

Evaluation of seven immunological assay reagents for hepatitis B virus surface antigen (HBsAg) in the sensitivity and the detection of HBsAg mutants

Mutants of hepatitis B virus surface antigen (HBsAg) are known as a cause of false-negative results in diagnostic tests for HBsAg; particularly when a diagnostic kit utilizes monoclonal antibodies to detect HBsAg. We compared seven HBsAg kits with regard to sensitivity for HBsAg subtypes (ad, ay) and their ability to detect nine different HBsAg mutants. Among them, the sensitivities of five kits were high and comparable to each other (0.2 - 0.3ng/ml). However, two kits were of lower sensitivity (0.8-1.3ng/ml, and 2.4-2.5ng/ml, respectively). Two kits, produced by the same company, reacted with all of the nine HBsAg mutants, but five kits showed false-negative results with one or more of the HBsAg mutants. These data indicate that there are differences in the detection sensitivities for HBsAg and abilities to detect HBsAg mutants among commercially available HBsAg kits, which may explain false-negative clinical results.     

Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg

We described a Hepatitis B surface antigen (HBsAg) subtyping method based on a commercial enzyme immunoassay (EIA) for detection of HBsAg in which the procedure was modified to include the use of monoclonal antibodies with restricted anti-HBs specificities. This method, which was able to classify HBsAg as: ayw1, ayw2, ayw3, ayw3 * (intermediate between ayw3 and ayw4 ), ayw4, ayr, adw2, adw4 and adr , was compared to counter electrophoresis procedure (CEP) by testing HBsAg positive sera from blood donors included in a prospective national epidemiological survey. Among the 256 HBsAg positive samples tested with both techniques, 111 (43.3%) could not be subtyped with CEP vs 10 (3.9%) with our modified EIA. This difference was related to the serum HBsAg concentration which must be greater than 3000 ng/mL and 100 ng/mL for CEP and EIA, respectively. The results obtained from 145 sera with both methods were concordant. Seventeen out of 18 samples partially classified as ay with CEP were completely determined with EIA.
This reliable procedure, derived from commercially available reagents, can be easily used in several applications such as large epidemiologic studies and as a substitute for nucleotide sequencing genotyping which is not adapted for large-scale screening and not applicable on samples from nonviremic hepatitis B virus (HBV) carriers.     

Persistence of HBsAg, HBeAg and Anti-HBe in Healthy Blood Donors

Abstract. The persistence of hepatitis B surface antigen (HBsAg), e antigen (HBeAg) and antibodies to e antigen (anti-HBe) was studied retrospectively in 197 Finnish voluntary blood donors, who had been found positive for HBsAg by routine screening in 1970 or 1971. Two samples were available from each donor: the first was taken in 1970 or 1971 and the second in 1978; the average interval between the specimens was 7.6 years. All except one (99.5%) of the donors remained HBsAg carriers during the whole study period. HBeAg was detected initially in 3 (2%) cases, all of whom lost HBeAg after the follow-up, and 2 converted to anti-HBe-positive. Anti-HBe was found in the first samples of 158 (80%) HBsAg carriers, 154 (97%) of whom were still positive for anti-HBe in the second samples. These results suggest that the HBsAg carrier stage is stable as for the presence of HBsAg and anti-HBe.     

Comparison between polyclonal and first and second generation monoclonal radioimmunoassays in the detection of hepatitis B surface antigen in patients with hepatocellular carcinoma

Serum from 221 black patients with hepatocellular carcinoma was tested for HBsAg using a polyclonal radioimmunoassay, and first and second generation monoclonal radioimmunoassays designated M1-RIA and M2-RIA. These monoclonal radioimmunoassays have a lower limit of detection of about 55 and 15 pg of HBsAg-associated epitopes per ml of serum. Correlations were made with other hepatitis B virus serologic markers such as anti-HBc and anti-HBs, using polyclonal radioimmunoassays. We found 47.5% (105/221) of the patients were HBsAg positive by conventional polyclonal radioimmunoassay; all of these patients were also reactive by monoclonal radioimmunoassay. Of the 116 polyclonal radioimmunoassay-negative patients, 4 (3.4%) were reactive by M1-RIA. These four patients were all positive for anti-HBc and/or anti-HBs antibodies. There were 106 of 112 patients negative for HBsAg by polyclonal radioimmunoassay and M1-RIA available for testing by the M2-RIA; 11 (10.4%) were found to be positive only by this test. Thus, with the use of M1- and M2-RIAs, the number of hepatocellular carcinoma patients negative for HBsAg by polyclonal radioimmunoassay was reduced by 14% in this population. More importantly, of the 11 M2-RIA-positive patients, six demonstrated anti-HBc and/or anti-HBs antibodies whereas the remaining five had no serologic evidence of recent or past hepatitis B virus exposure. Finally, of the 21 patients in this series with no markers of hepatitis B virus infection using polyclonal radioimmunoassay and M1-RIA, five (24%) were reactive for HBsAg-associated epitopes by M2-RIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatitis δ virus attaches to human hepatocytes via human liver endonexin II, a specific HBsAg binding protein

SUMMARY. Recently we identified human liver endonexin II (EII) as a specific hepatitis B surface antigen (HBsAg) binding protein. To investigate whether EII is also able to interact with the HBsAg envelope of the hepatitis δ virus (HδV). immunoprecipitation experiments were performed. HδV particles could be co-precipitated by polyclonal rabbit anti-EII, but not by rabbit anti-glutathiontransferase (GSTφ) antibodies from an HδV-enriched fraction containing EII or GSTφ. These findings suggest that HδV particles were co-precipitated by anti-EII as a consequence of the binding between HBsAg present in the HδV envelope and EII. Furthermore, binding of HδV particles to human hepatocytes could be inhibited by incubation of the liver cells with rabbit anti-EII IgG or the HδV particles with anti-idiotypic (anti-HBsAg) antibodies, developed spontaneously in rabbits immunized with EII. These findings support the assumption that small HBsAg present in the HδV envelope is important for the attachment to the hepatocytes and that EII plays an important role in this process.     

HBsAg-serum protein complexes stimulate immune T lymphocytes more efficiently than do pure HBsAg

HBsAg from plasma of chronic hepatitis B carriers was purified by affinity chromatography using a mouse monoclonal antibody specific for HBsAg. Elution with buffer at two different pH values separated HBsAg into two fractions: one contained high amounts of immune complexes associated with HBsAg; the other contained larger quantities of the HBsAg polypeptides P24 and GP27 and only small amounts of immunoglobulin. When compared for effects on stimulating the proliferative response of freshly isolated lymphocytes and an HBsAg-specific T cell clone, the HBsAg fraction containing a high proportion of immunoglobulin was much more potent than HBsAg with low amounts of immunoglobulins or pure HBsAg, which was isolated from the culture supernatant of the human hepatoma cell line (PLC/PRF/5). The plasma-derived HBsAg with low amounts of complexed immunoglobulins became more immunogenic in the presence of an anti-HBsAg monoclonal IgG. The present results, combined with earlier findings, suggest that HBsAg associated with immune complexes is a more potent stimulator of T cells than purer HBsAg preparations due to an increase in the efficiency of monocytes to capture the antigen through binding to immune complexes for subsequent processing and presentation of the antigen. These observations could be of relevance for the preparation of effective hepatitis B vaccines from recombinant DNA and peptide synthesis technologies.     

Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees

Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10(-10) mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.     

Hepatitis B virus vaccine: identification of HBsAg/a and HBsAg/d but not HBsAg/y subtype antigenic determinants on a synthetic immunogenic peptide.

On the basis of theoretical considerations, a peptide (H peptide) was synthesized by Hopp and Woods [Hopp, T. P. & Woods, K. R. (1981) Proc. Natl. Acad. Sci. USA 78, 3824---3828]. This peptide contains a sequence of six amino acids postulated to represent the major epitope, or antibody-combining site, of hepatitis B virus surface antigen (HBsAg). We have used passive hemagglutination inhibition with monospecific antibodies against the a, d, and y subdeterminants of this antigen and against human serum albumin to investigate the antigenic specificities on this peptide, and we have found it to contain the HBsAg/a and HBsAg/d but not HBsAg/y or human serum albumin subdeterminants. When the peptide was conjugated onto human erythrocytes and injected into mice, it induced the formation of anti-HBsAg with and without the use of Freund's adjuvant. If anti-HBsAg/a confers immunity to infection with hepatitis B virus, as is generally thought, these findings may permit the development of a synthetic vaccine lacking all unnecessary antigenic determinants.     

Hepatitis C virus infection in anti-HBe-positive HBsAg carriers with chronic liver disease.

In the present study, sera from chronic hepatitis B surface antigen (HBsAg) carriers positive for antibody to hepatitis B 'e' antigen (anti-HBe) with evolutive liver disease as correlated with anti-HBe-positive healthy carriers, were examined for antibodies to hepatitis C virus (HCV). Anti-HCV antibodies were detected in 32/124 (25.8%) anti-HBe-positive carriers with chronic liver disease and in none of the 46 healthy carriers. When anti-HCV positivity was evaluated in relationship to the degree of severity of liver disease and possible confounding factors such as hepatitis B virus replication or other potential hepatolesive factors were eliminated by using logistic regression, the odds ratio of liver cirrhosis versus chronic persistent hepatitis was 18 (95%, CI 3.5-92.5). Therefore, our results indicate that HCV may be implicated in the determinism and severity of liver damage in a significant proportion of anti-HBe-positive chronic HBsAg carriers.     

Identification of a hepatitis B virus S gene mutant in lamivudine-treated patients experiencing HBsAg seroclearance

BACKGROUND & AIMS: Seroclearance of hepatitis B virus (HBV) surface antigen (HBsAg) is a rare event in chronic hepatitis B patients receiving lamivudine therapy. It is generally believed to be a benevolent sign, implicating clearance of viremia. The aim of this study is to examine the authenticity of this dogma. METHODS: In a 5-year period, 11 patients treated with lamivudine experienced seroclearance of HBsAg. The clinical data were examined. The HBV S gene sequences derived from the patient's serum samples before and after seroclearance of HBsAg were analyzed. RESULTS: Serum HBV-DNA could be detected by nested polymerase chain reaction (PCR) in all 11 patients, by 1-step PCR in 8, and by Cobas Amplicor HBV-DNA test (>200 copies/mL) in 5. A mutation hot spot, P120A in the S gene, was identified in 6 of the 11 patients. Site-directed mutagenesis experiments indicated that the Ausria-II RIA test failed to detect this mutant. Decreased sensitivity of detection was also observed when other monoclonal antibodies were applied. CONCLUSIONS: Seroclearance of HBsAg during lamivudine therapy may not indicate viral clearance. Specifically, it may be caused by a point mutation in the S gene, which results in detection failure. In such patients, further verification and follow-up using a sensitive HBV-DNA test are advised.     

High affinity monoclonal antibodies to hepatitis B surface antigen (HBsAg) produced by somatic cell hybrids

Somatic cell hybrids (hybridomas) have been established which produced antibodies to hepatitis B surface antigen (HBsAg) by immunizing BALB/c/mice with a highly purified preparation of HBsAg and fusing their splenocytes with the myeloma cell line P3-NSI/1-Ag4-1. The route of immunization, interval between primary and secondary immunizations, and immunizing antigen concentration appeared crucial for optimal establishment of the hybrid cell lines. From one cell fusion, described here in detail, we established 47 hybrid cell lines secreting antibody to HBsAg (anti-HBs). The resultant hybrids produced anti-HBs of sufficient affinity and concentration to yield values over 400 times background in a solid-phase radioimmunoassay. Three of the secreting hybrids have been cloned by dilutional techniques. The anti-HBs from such hybrids showed specificity for antigens present on HBsAg subtypes (adw and ayw). Monoclonal anti-HBs antibodies to various antigenic components of HBsAg may lead to refinement in the immunodiagnosis of hepatitis B infection and serve as potent probes in the characterization of this complex particle. Moreover, they offer the potential for the development of highly specific immunoprophylactic reagents.     

己酮可可碱联合阿苯达唑治疗小鼠泡球蚴病的疗效观察

目的 观察己酮可可碱（PTX）和阿苯达唑（ABZ）单独及联合用药对小鼠继发性泡球蚴病的疗效。 方法 对小鼠继发性泡球蚴病进行药物治疗，各治疗组药物用量分别为：ABZ组 50 mg/（kg·d）；PTX高剂量组360 mg/（kg·d）；PTX低剂量组180 mg/（kg·d）；联合组 ABZ 50 mg/（kg·d） + PTX 180 mg/（kg·d）；感染对照组（未治疗组）和空白对照组均给予等体积生理盐水，用小鼠灌胃针经口每天灌胃给药1次，连续治疗100 d后 （其间14只死亡），检测各小鼠泡球蚴湿重、抑囊率及小鼠血清细胞因子转化生长因子?鄄β （TGF-β）、白细胞介素?鄄2 （IL-2）和IL?鄄10；并对泡球蚴组织进行病理组织学和超微结构观察。 结果 PTX在体外能有效地杀灭原头节（高剂量组为100%）, 在体内对泡球蚴抑制作用虽较弱（高剂量组为37%），但能增强小鼠的免疫力。联合用药对泡球蚴有明显的抑制作用，抑囊率为88 %，ABZ抑囊率为58 % （P<0.05）。 结论 PTX联合ABZ治疗小鼠继发性泡球蚴病疗效明显优于ABZ。     

Selecting binding and complement-mediated lysis of human hepatoma cells (PLC/PRF/5) in culture by monoclonal antibodies to hepatitis B surface antigen.

High-affinity 125I-labelled monoclonal antibodies to hepatitis B virus surface antigen (HBsAg) bind to human hepatocellular carcinoma cell line, PLC/PRF/5, which synthesizes and secretes HBsAg. These monoclonal antibodies of the IgG1, IgG2a, and IgM isotypes are directed against different antigenic determinants on HBsAg and, in the presence of complement, both anti-HBs IgG2a and IgM, but not anti-HBs IgG1, lyse PLC/PRF/5 cells in culture. Although there is a low level of anti-HBs binding (especially with anti-HBs IgM) to human hepatoma cell lines which do not synthesize HBsAg (SK-Hep 1 and Mahlavu cells), this interaction does not lead to complement-mediated cell lysis and is thought to be nonspecific. Minimal binding of 125I-labeled anti-influenza HA antigen IgM binding to PLC/PRF/5 cells was also detected, but this likewise did not lead to complement-mediated cell lysis. These results indicate that a human hepatocellular carcinoma cell line, persistently infected with hepatitis B virus, can be recognized and lysed by monoclonal antibodies directed against specific determinants of HBsAg. Monoclonal antibodies to this viral envelope protein may prove to be useful immunodiagnostic and immunotherapeutic agents when such viral epitopes are expressed on the surface of infected cells.     

Regulation of the human immune response to HBsAg: effects of antibodies and antigen conformation in the stimulation of helper T cells by HBsAg

The role of accessory cells (antigen-presenting cells) in binding HBsAg in the response of human T cells to this antigen was studied. Antibodies to HBsAg of IgG class increased significantly the amount of HBsAg that was captured and internalized by accessory cells in vitro. On the other hand, antibodies to HBsAg of IgM class or the F(ab')2 and Fab fragments of antibodies to HBsAg of IgG class did not modify the amount of HBsAg associated to these cells. HBsAg that was subjected to various denaturing treatments (acid, organic solvents, urea and heat) was compared for its capacity to react with antibody to HBsAg and stimulate the response of helper T lymphocytes. Results presented here indicate that HBsAg denatured by treatment with formic acid was captured by accessory cells and presented to the T cells much more efficiently than the native HBsAg. These results suggest that the response of helper T lymphocytes to some antigens such as HBsAg can be affected greatly by the presence of antibodies or the antigens' conformation.     

Identification of human megakaryocyte coagulation factor V

Specific monoclonal and polyclonal antibody reagents and a double antigen indirect immunofluorescence microscopy technique were used to visualize coagulation factor V in human bone marrow. Marrow aspirates were smeared directly on glass slides, or washed and cytospun onto glass slides, or processed and plated into a plasma/methylcellulose cell culture system. Morphologically identifiable colonies of megakaryocytes, erythrocytes, granulocytes, or monocytes/macrophages were removed from 14- to 18-day marrow culture dishes by micropipette and streaked onto glass slides. Smears of marrow cell preparations were air-dried, fixed, washed, and incubated sequentially with primary IgG antibody reagents and with secondary anti-IgG antibody reagents conjugated with either fluorescein or rhodamine. Preparations were examined and photographed through a microscope suitably equipped for two-color fluorescence and phase contrast analysis. Cells of megakaryocytic lineage were identified by their immunofluorescent reactivity with murine monoclonal antibody HP1-1D, specific for human platelet plasma membrane glycoprotein IIb/IIIa (GP IIb/IIIa), or by their immunofluorescent reactivity with monoclonal or polyclonal antibodies specific for von Willebrand factor (vWF) or for platelet factor 4 (PF4). Coagulation factor V in bone marrow was detected by simultaneous immunofluorescent staining with polyclonal burro anti-human factor V antibody or with a panel of murine monoclonal anti-human factor V antibodies. The double antigen immunofluorescence staining technique, incorporating appropriate controls, revealed that coagulation factor V was principally located in marrow cells simultaneously identified as megakaryocytes by antibodies to GP IIb/IIIa, vWF, or PF4. The specific immunofluorescence of factor V in megakaryocytes and platelets was eliminated when excess purified factor V antigen was preincubated with anti-factor V antibody. Our observations establish the presence of human megakaryocyte coagulation factor V, confirm the presence of human platelet factor V, and indicate that human megakaryocyte/platelet coagulation factor V is a lineage-associated protein.     

Genetic alterations in the gene encoding the major HBsAg: DNA and immunological analysis of recurrent HBsAg derived from monoclonal antibody-treated liver transplant patients.

A gene region encoding a segment of the major surface protein, HBsAg, of hepatitis B virus was analyzed from serum samples after orthotopic liver transplantation of three hepatitis B virus chronic carrier patients treated with a human anti-hepatitis B virus monoclonal antibody (SDZ OST 577). Each of these three patients became HBsAg negative after transplantation and therapy with the human anti-hepatitis B virus monoclonal antibody but returned to HBsAg positivity (first detected 143,251 and 252 days after the transplantation). Polymerase chain reaction DNA amplification was performed on DNA from serum samples showing low levels of recurrent HBsAg and reduced antigen reactivity with SDZ OST 577 antibody. Polymerase chain reaction DNA included a 230-bp highly conserved, major S gene region that was cloned into M13 bacteriophage; analysis of this DNA segment provided a consensus of DNA sequences for the serum samples exhibiting altered reactivity with the therapeutic monoclonal. Analysis of independent DNA clones from serum samples of patients exhibiting low but detectable recurrent serum levels of posttherapy HBsAg revealed the presence of S protein variant sequences when compared with polymerase chain reaction DNA derived from the original infected liver or pretherapy serum HBsAg. Genetic variation was predominant in a highly conserved peptide domain that has previously been implicated in antibody binding and neutralizing antibody epitopes. In independent patients infected with either adw or ayw hepatitis B virus subtypes, single nucleotide changes resulted in one to two amino acid differences for each variant allele (residues 124, 129, 131, 137, 140 and/or 145) when compared with pretherapy viral DNA. Administration of serum containing one of these variant viruses to a single hepatitis B-naive chimpanzee resulted in subclinical hepatitis and detectable levels of circulating anti-HBs and anti-HBc antibodies 49 and 70 days after virus administration, respectively. Hepatitis B virus DNA was recovered on liver biopsy between 6 and 8 wk after inoculation, although the animal remained persistently seronegative for HBsAg. DNA sequence analysis of both primate and patient liver hepatitis B virus confirmed the presence of the DNA encoding the S protein variant and associates this DNA with the predominant hepatotropic virus in liver infection.     

Hepatitis C virus infection, HBsAg carrier state and hepatocellular carcinoma: relative risk and population attributable risk from a case-control study in Italy.

In 1990, a case-control study was conducted in Italy to investigate the possible association between HCV infection and hepatocellular carcinoma (HCC). Serum samples from 65 subjects with newly diagnosed hepatocellular carcinoma and 99 hospital control subjects were tested for the presence of anti-HCV by second-generation ELISA test; positive sera were assayed by RIBA anti-HCV second-generation test. In addition, samples were tested for hepatitis B surface antigen (HBsAg), antibodies to the hepatitis B core antigen (anti-HBc), and antibodies to HBsAg (anti-HBs). The presence of HCV and/or HBsAg serologic markers was significantly associated with hepatocellular carcinoma risk: the relative risk (RR) of HCC was 21.3 (95% CI = 8.8-51.5) for anti-HCV positivity in the absence of HBsAg; the relative risk of HCC was 13.3 (95% CI = 5.5-32.2) for the presence of HBsAg in the absence of anti-HCV. A higher risk (77.0) was observed when both markers were present. These findings indicate that HCV and HBsAg are independent risk factors for HCC. The results of multivariate analysis showed that the adjusted RR linking anti-HCV and HCC was 26.9 (95% CI = 9.9-72.5), the adjusted RR linking HBsAg and HCC was 11.4 (95% CI = 3.1-41.4), whereas no association (RR 1.5; 95% CI = 0.6-3.6) was found to link HCC with anti-HBc and/or anti-HBs positivity. Through the computation of population attributable risk we estimate that 25% of HCC cases occurring in Italy could be attributed to anti-HCV positivity alone and 20% to HBsAg carrier state alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

提高酶联免疫分析法检测乙型肝炎表面抗原诊断试剂质量的几个重要因素

本文报道以单克隆或多克隆抗体不同搭配用于包被和标记的ELISA比较检测HBsAg。初步探讨了影响ELISA敏感性、特异性和稳定性的一些重要因素。结果显示,S/N值随包被和标记抗体而异,可见抗体选择重要。由于目前常用的辣根过氧化酶法和改良的辣根过氧化酶法都相当程度地导致抗体活性下降,因此在选择抗体时,标酶抗体活性、亲和力更应受到重视。应用单克隆抗体在提高检测敏感性、特异性方面优点突出。ELISA阴性(和阳性)对照的选择应具有人群代表性,以便在此基础上,结合试验的程序、试剂的质量确定正确的阈值。     

Reactivation of resolved hepatitis B virus infection after immunosuppression: Is it time to adopt pre-emptive therapy?

New therapeutic options like monoclonal antibodies (anti-CD20/rituximab) and hematopoietic stem cell transplantation (HSCT) have increased both the effectiveness of therapies and the risk for reactivation of Hepatitis B virus (HBV). We describe two cases with serological evidence of resolved HBV infection (hepatitis B surface antigen (HBsAg) negative/antibody to hepatitis B core antigen (anti-HBc) and antibody to hepatitis B surface antigen (anti-HBs) positive), who developed reverse seroconversion (clearance of HBsAb/appearance of HBsAg) with active HBV infection after treatment with combination of conventional chemotherapy, rituximab and autologous HSCT for hematological malignancies. Review of the literature highlights the increasing incidence of HBV reactivation in patients with resolved infection and raises concerns as to whether current guidelines for pre-chemotherapy screening with sensitive HBV-DNA assays and serial monitoring for anti-HBs titres should be implemented also for patients with resolved infection. Future studies should aim at clarifying the cost-benefit from administration of nucleoside analogues in these patients.