Organ, tissue, and cell transplantations expose recipients

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1 GASTROENTEROLOGY 2008;135: CLINICAL LIVER, BILIARY TRACT Efficacy of Serologic Marker Screening in Identifying Hepatitis B Virus Infection in Organ, Tissue, and Cell Donors DOMINIQUE CHALLINE,*,, STÉPHANE CHEVALIEZ,*, and JEAN MICHEL PAWLOTSKY*,, *French National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, and the Viral Emergency and Organ, Tissue and Cell Donor Screening Laboratory, Hôpital Henri Mondor, Université Paris 12, Créteil, France; and INSERM U841, Créteil, France See editorial on page Background & Aims: Screens for serologic markers of hepatitis B virus (HBV) are used to prevent its transmission through transplantation. However, exclusion of noninfectious seropositive donors exacerbates graft shortages, and a residual risk of transmission by seronegative donors also exists. This study assessed the risk of HBV associated with different HBV serologic profiles in organ, tissue, and cell transplants, as well as the risk of HBV transmission from seronegative donors. Methods: A total of 11,155 consecutive organ, tissue, and cell donors were screened for HBV serologic markers. HBV DNA was screened for in 626 donors with at least one HBV serologic marker and 1433 multiple organ donors who were HBV seronegative or had anti-hepatitis B surface antigens (HBs) antibodies alone. Results: HBV DNA was detected in most HBs-antigen positive donors, but HBV-DNA levels were considerably lower than in patients with chronic hepatitis B. HBV DNA also was found in organ and cornea donors without HBs antigen. The prevalence of HBV DNA in organ donors with no HBV serologic markers or isolated anti-hbs antibodies was 0.07% (95% confidence interval, 0.01% 0.40%). One HBV-DNA positive organ donor with isolated anti-hbs antibodies had amino acid substitutions in the hepatitis B surface antigen sequence. Conclusions: The analytic sensitivity of commercial hepatitis B surface antigen assays and their ability to detect HBsAg mutants should be improved. The utility and cost-efficacy ratio of systematic HBV-DNA testing should be assessed with the goal of excluding HBV-DNA positive donations not identified through serologic testing while retaining donations that carry no risk of HBV transmission. Organ, tissue, and cell transplantations expose recipients to various viral agents. Hepatitis B virus (HBV) infection is highly prevalent worldwide, including in countries with active transplantation programs. 1 De novo HBV infection has been reported in recipients of organs, tissues (including corneas), and cells from infected donors. 2 6 HBV infection is more likely to become chronic in this setting, and the chronic infection has a more severe outcome than in immunocompetent patients. Prevention of viral transmission during transplantation is based primarily on the donor screening for serologic markers of infection. 2 This approach has 2 major drawbacks, however, namely exclusion of noninfectious seropositive donors, exacerbating the ongoing organ and tissue shortage; and a residual risk of transmission by seronegative donors. Donors blood is screened systematically for hepatitis B surface antigen (HBsAg) and anti hepatitis B core (HBc) antibodies in most countries, and for anti-hbs antibodies in some countries such as France. Donors with HBsAg and/or anti-hbc antibodies are excluded from donation. Indeed, most donors with detectable HBsAg can transmit HBV. Donors with isolated anti-hbc antibodies and active viral replication also can transmit HBV infection to organ, tissue, and cell recipients. However, the risk of transmission is only high in liver recipients, 7 11 meaning that, in a context of graft shortage, such donors might conceivably be used in other transplantation settings. The utility of anti-hbs antibody testing of anti-hbc positive donors for this purpose is questionable because donors with both markers (the recovery profile) have been known to transmit HBV to liver recipients Finally, false-positive and falsenegative serologic results are frequent in cadaveric donors of corneas, which are also in short supply. 12 Abbreviation used in this paper: PCR, polymerase chain reaction by the AGA Institute /08/$34.00 doi: /j.gastro

2 1186 CHALLINE ET AL GASTROENTEROLOGY Vol. 135, No. 4 Table 1. Serologic Profiles of Organ, Tissue, and Cell Donors From Group A N n (%) HBsAg ( ) (%) Anti-HBc Ab ( ) and anti-hbs Ab ( ) (%) Anti-HBc Ab ( ) alone (%) HBsAg ( ) and anti-hbs Ab ( ) (%) Organ donors (12.2) 20 (1.2) 121 (7.4) 53 (3.2) 5 (0.3) Tissue donors (4.9) 2 (0.1) 130 (3.9) 33 (1.0) 0 Stem cell donors (4.1) 5 (0.3) 61 (3.3) 9 (0.5) 0 Cord blood donors (1.7) 2 (0.1) 44 (1.3) 10 (0.3) 0 Cornea donors (13.7) 8 (0.9) 62 (7.2) 21 (2.4) 27 (3.1) Living organ donors (7.1) 0 10 (5.5) 3 (1.6) 0 Total 11, (5.6) 37 (0.3) 428 (3.8) 129 (1.2) 32 (0.3) NOTE. These donors were found to have at least 1 of the 3 HBV serologic markers, namely HBsAg, anti-hbc antibodies (Ab), and anti-hbs antibodies (vaccination profiles, characterized by the presence of anti-hbs antibodies alone, were excluded). N, total number of tested donors; n, number of donors with at least 1 HBV serologic marker (excluding anti-hbs antibodies alone). The prevalence of each serologic profile has been calculated out of N (percentages are shown in parenthesis). The residual risk of HBV transmission during transplantation of organs, tissues, and cells from HBV-seronegative donors (ie, HBsAg-negative, anti-hbc negative) is unknown. The probability of HBV viremia being detectable in HBV-seronegative donors at the time of tissue donation recently was estimated to be about 1 in 34,000 in the United States, based on the incidence-windowperiod model; this represents less than one viremic donor per year. 13 However, no such data are available for organ transplantation, a setting in which the recipient is exposed to a substantially higher risk of viral transmission than during tissue and cell transplantation. Indeed, the prevalence of chronic viral infections, including HBV, human immunodeficiency virus, and hepatitis C virus (HCV) infection, is significantly higher among organ donors than in the general population and in tissue and cell donors in the same geographic region (Challine et al, unpublished data). 14 In addition, HCV RNA has been detected in organ and tissue donors with no detectable anti-hcv antibodies, and HCV transmission to organ and tissue recipients has been shown to occur. 15,16 Whether this also applies to HBV is not known. The goals of this study, involving a large population of organ, tissue, and cell donors, were to assess the following: (1) the risk of HBV transmission associated with different HBV serologic profiles and the utility of testing these markers for preventing this risk, and (2) the risk of HBV transmission from seronegative donors, as well as the underlying virologic mechanisms. Patients and Methods Donors Between May 2000 and May 2004, there were 11,155 consecutive organ, tissue, and cell donors tested in the Viral Emergency and Organ, Tissue and Cell Donor Screening Laboratory of Henri Mondor Hospital, University of Paris 12, for viral markers, before the grafts could be released. Tissue donors included bone, tendon, fascia, skin, valve, artery, and vein donors. Donor blood samples originated from public hospitals and tissue and cell banks in the Paris area. The samples were centrifuged on receipt and tested for regulatory viral markers, including 3 HBV markers (HBsAg, anti-hbc, and anti-hbs), anti human immunodeficiency virus antibodies, human immunodeficiency virus p24 antigen, anti-human T-cell leukemia virus I and II antibodies, anti-hcv antibodies, and, in organ and cell donors only, anticytomegalovirus antibodies and anti Epstein Barr virus antibodies. Serologic testing was performed urgently for brain-dead mul- Table 2. Prevalence of Detectable HBV DNA and Mean ( SD) HBV-DNA Levels in Organ, Tissue, and Cell Donors From Group A HBsAg ( ) Anti-HBc Ab ( ) anti-hbs Ab ( ) Donors N n HBV DNA ( ) Mean SD (range) HBV-DNA level, log 10 IU/mL n HBV DNA ( ) Mean SD (range) HBV-DNA level, log 10 IU/mL Organ donors (85.0%) ( ) NA Tissue donors (100%) ( ) NA Stem cell donors (60.0%) ( ) 61 0 NA Cord blood donors (100%) ( ) 44 0 NA Cornea donors (12.5%) (4.8%) ( ) Living organ NA 10 0 NA donors Total (67.5%) ( ) (.7%) ( ) NOTE. These donors were found to have at least 1 HBV serologic marker, among HBsAg, anti-hbc antibodies (Ab), and anti-hbs antibodies (vaccination profiles, characterized by the presence of anti-hbs antibodies alone, were excluded). The results are presented according to the type of donor. NA, not applicable; N, total number of donors with at least 1 HBV serologic marker; n, number of donors with each different serologic profile.

3 October 2008 HBV REPLICATION IN GRAFT DONORS 1187 tiple organ donors, and within hours for living donors of tissues and cells. The samples then were stored at 80 C until use in this study. Excluding donors with anti-hbs alone (vaccination profile), 626 donors (5.6%) had at least 1 of the 3 HBV serologic markers. These donors (study group A) included 199 brain-dead, heart-beating multiple organ donors, 165 tissue donors, 75 stem cell donors, 56 cord blood donors, 118 cadaveric cornea donors, and 13 living organ donors (blood samples from cadaveric cornea donors had been collected at various times after death and were often of poor quality) (Table 1). Among these 626 donors, 69 (0.6%) were HBsAg-positive, including 32 who had both HBsAg and anti-hbs antibodies, 428 (3.8%) were positive for both anti-hbc and anti-hbs antibodies, and 129 (1.2%) had isolated anti-hbc antibodies. Among the 32 donors who had both HBsAg and anti-hbs antibodies, 14 also had anti-hbc antibodies (4 organ donors and 10 cornea donors), whereas 18 did not (1 organ donor and 17 cornea donors) (Table 1). Study group B comprised the 1433 consecutive braindead, heart-beating multiple organ donors tested during the study period who were negative for all 3 HBV serologic markers (n 912) or who had a vaccination profile (anti-hbs alone; n 521). Methods Serologic testing. All the donors initially had been screened for the 3 HBV serologic markers by means of Vitros ECi HBsAg (detection limit, 0.16 ng/ml), Vitros ECi anti-hbc, and Vitros ECi anti-hbs assays (Ortho-Clinical Diagnostics, Raritan, NJ). All samples from groups A and B were retested systematically for HBsAg by means of 2 additional assays: Architect HBsAg (detection limit, ng/ml) (Abbott Diagnostic, Chicago, IL) and Vidas HBsAg Ultra (detection limit, 0.12 ng/ml) (BioMérieux, Marcy l Etoile, France). The presence of HBsAg in HBsAg-positive samples was confirmed by neutralization. Antibodies against hepatitis delta virus (HDV) were sought in group A donors by means of the ETI-AB-DELTAK-2 enzyme immunoassay (DiaSorin, Saluggia, Italy). HBV-DNA detection and quantification. HBV DNA was screened for and quantified in the 626 group A samples and the 1433 group B samples by means of the Cobas TaqMan HBV standardized real-time polymerase chain reaction (PCR) assay (Roche Molecular Systems, Pleasanton, CA) after manual extraction with the QiaAmp DNA minikit (Qiagen, Hilden, Germany) from 0.5 ml of serum or plasma. The results were expressed in international units/milliliter or log 10 international units/milliliter. The assay has a detection limit of 6 HBV DNA IU/mL, and a dynamic range of quantification from 6 to 10 8 IU/mL. All samples with a viral load of less than 100 IU/mL in the initial determination were retested systematically using the same procedure. Determination of the pres-s and prec-c gene sequences. The full-length nucleotide and deduced amino acid sequences of the pres1 pres2-s HBV gene were determined in all group A and group B patients with detectable HBV DNA, using a previously published method. 17 The prec-c gene sequence was determined in donors positive for HBV DNA but negative for anti-hbc antibodies as described elsewhere. 17 Nucleotide sequence accession numbers. Sequence data from this article have been deposited in GenBank under accession numbers EU to EU Results Prevalence of HBV DNA in Organ, Tissue, and Cell Donors With at Least One HBV Serologic Marker (Excluding Isolated Anti-HBs Positivity) Table 2 shows the prevalence of HBV DNA and the mean ( SD) HBV-DNA levels in the 626 organ, tissue, and cell donors in group A, who all had at least 1 of the 3 HBV serologic markers (excluding anti-hbs Table 2. Continued Anti-HBc Ab ( ) alone HBsAg ( ) anti-hbs Ab ( ) n HBV DNA ( ) Mean SD (range) HBV- DNA level, log 10 IU/mL n HBV DNA ( ) Mean SD (range) HBV-DNA level, log 10 IU/mL 53 2 (3.8%) ( ) 5 2 (40.0%) ( ) 33 0 NA 0 0 NA 9 0 NA 0 0 NA 10 0 NA 0 0 NA 21 2 (9.5%) ( ) 27 1 (3.7%) NA 0 0 NA (3.1%) ( ) 32 3 (9.4%) ( )

4 1188 CHALLINE ET AL GASTROENTEROLOGY Vol. 135, No. 4 alone), according to the type of donation and the HBV serologic profile. HBsAg-positive donors. As shown in Table 2, the vast majority of HBsAg-positive, brain-dead, heart-beating organ donors and living organ, tissue, stem cell, and cord blood donors with detectable HBsAg also were HBV- DNA positive. In contrast, only 1 of 8 HBsAg-positive cornea donors who had been sampled several hours after death was found to be HBV-DNA positive, suggesting false-positive HBsAg detection in this subpopulation. Indeed, the sera of the 7 HBV-DNA negative cornea donors looked abnormal (ie, hemolyzed, icteric, or cloudy); their HBsAg test was weakly reactive and anti-hbc antibodies were not detected. None of the HBsAg-positive donors was positive for anti-hdv antibodies. All organs, tissues, and cells from HBsAg-positive donors were excluded from transplantation. Anti-HBc and anti-hbs dual-positive donors. Negative HBsAg detection was confirmed with the 2 additional HBsAg assays in all of these donors. None of the brain-dead, heart-beating organ donors and none of the living donors of organs, tissues, stem cells, or cord blood who were simultaneously positive for anti-hbc and anti- HBs antibodies (resolved HBV infection profile) was HBV-DNA positive. In contrast, 3 (4.8%) of 62 cadaveric cornea donors were HBV-DNA positive. This suggested false-negative detection of HBsAg and false-positive detection of anti-hbs antibodies in these donors, all of whose sera looked abnormal. The corneas from the HBV- DNA positive donors were not used because of indeterminate serologic results for other viruses. Two donors (1 organ and 1 tissue donor) with both anti-hbc and anti- HBs antibodies but without detectable HBV DNA were anti-hdv positive. Donors with isolated anti-hbc antibodies. Negative HBsAg detection was confirmed with the 2 additional HBsAg assays in all cases. As shown in Table 2, 129 donors were positive for anti-hbc alone, including 53 brain-dead organ donors, 3 living organ donors, 33 tissue donors, 9 stem cell donors, 10 cord blood donors, and 21 cornea donors. HBV DNA was detected in 2 brain-dead, heart-beating organ donors (3.8%) and in 2 cornea donors (9.5%). The liver of 1 of the 2 HBV-DNA positive organ donors was transplanted to a patient with HBVrelated end-stage liver disease. This patient had to be retransplanted 21 days after the first graft. No other organs or tissues from this donor were transplanted. No relevant information was available on the second organ donor. The corneas from the 2 HBV-DNA positive donors were not used because of indeterminate serologic results for other viruses. HBsAg-positive and anti-hbs antibody positive donors. Twenty-seven of the 32 donors with both HBsAg and anti-hbs were cadaveric cornea donors. One of them also was positive for anti-hdv antibodies. Among the cornea donors with both HBsAg and anti- HBs antibodies, only 1 (3.7%) was HBV-DNA positive. He also had detectable anti-hbc antibodies and the corneas were not used. Two brain-dead, heart-beating organ donors out of 5 with both HBsAg and anti-hbs antibodies were found to be HBV-DNA positive. Both of them also were anti-hbc antibody positive. They were excluded from donation. Whatever the serologic profile, when HBV DNA was present its level was generally low, on average log 10 IU/mL. HBV-DNA levels are shown according to the serologic profile and type of donor in Table 2. Prevalence of HBV DNA in Brain-Dead, Heart-Beating Organ Donors With No HBV Serologic Markers or With Isolated Anti-HBs Antibodies HBV DNA was sought in 1433 consecutive braindead, heart-beating multiple organ and tissue donors with no HBV serologic markers or with isolated anti-hbs antibodies (group B). HBV DNA was found in only 1 of them (prevalence, 0.07%; 95% confidence interval, 0.01% 0.40%). This donor was among the 521 donors with isolated anti-hbs antibodies, whereas HBV DNA was not found in any of the 912 donors with no HBV serologic markers. The HBV-DNA positive donor was repeatedly found to be HBsAg and anti-hbs antibody negative on retesting. The HBV-DNA level in this donor was 3.0 log 10 IU/mL. The liver and kidneys from this donor were transplanted. The liver recipient was transplanted for HBVrelated end-stage liver disease and died 2 months later. One kidney recipient was HBsAg-negative before transplantation. He lost the graft on day 5 and was retransplanted 3 years later. He had the same HBV serologic profile as the first donor at the time of the second transplant (HBsAg-negative, anti-hbc negative, and anti- HBs positive), but HBV-DNA testing was not performed and the patient died 5 months after the second transplant. Finally, the second kidney graft was lost on day 7 and no serologic information on the recipient was available. Full-Length pres1 pres2-s Sequence Analysis We attempted to determine the full-length nucleotide and deduced amino acid sequences of the pres1 pres2-s gene in all HBV-DNA positive donors. Among HBV-DNA positive donors, PCR amplification of the full-length pres1 pres2-s region was possible in 23 of the 25 HBsAg-positive donors, 2 of the 4 donors with isolated anti-hbc antibodies, and all 3 donors with both HBsAg and anti-hbs antibodies. Low HBV- DNA levels were the most likely reason why PCR failed to amplify this long fragment in the remaining 7 HBV-DNA positive donors. Among the donors in whom the pres1 pres2-s region could be amplified, 12 were infected with HBV genotype D, 7 with genotype A, 3 with genotype C, 3 with genotype E, 2 with genotype B, and 1 with genotype G. The pres1 pres2-s

5 October 2008 HBV REPLICATION IN GRAFT DONORS 1189 Figure 1. Amino acid sequence alignment of the major hydrophilic region (in squares) HBsAg in the 29 donors in whom the full-length pres1 pres2-s gene could be amplified, including 23 donors with detectable HBsAg, 2 donors with isolated anti-hbc antibodies, 3 donors with both HBsAg and anti-hbs antibodies, and the donor with isolated anti-hbs antibodies. The a determinant sequence (amino acids ) is underlined at the top. The sequences are aligned relative to an HBV genotype A prototype sequence, together with prototype sequences of genotypes A H. The donors genotypes are indicated in parentheses. Amino acid positions previously reported to bear substitutions associated with HBsAg detection failure in HBV-DNA positive patients are shown in gray. Amino acid substitutions observed at these positions in the donors of this study are shown in bold letters. region also could be PCR-amplified in the HBV-DNA positive brain-dead, heart-beating organ donor with isolated anti-hbs antibodies. This donor was infected with HBV genotype D. Figure 1 shows an alignment of the sequences of the major hydrophilic region of HBsAg, that contains the a determinant of HBsAg, an immunodominant neutralizing epitope spanning amino acids , 18,19 in the 29 HBV- DNA positive donors in whom the full-length pres1 pres2-s region could be amplified, according to their serologic profile. Amino acid substitutions within and without the a determinant have been linked to HBsAg detection failure in patients with chronic HBV infection. 20,21 Their positions are indicated in gray in Figure 1. Among the 23 HBsAg-positive donors, 3 harbored amino acid substitutions within or close to the a determinant known to alter the antigenic and immunologic structure of HBsAg: donor 3 at position 159, donor 4 at position 133, and donor 6 at positions 129 and 160 (Figure 1). However, HBsAg was detected in all of these donors with all the immunoenzymatic assays used. One donor with both HBsAg and anti-hbs antibodies (donor 27) had a Thr to Ala substitution at position 131 of the a determinant (T131A) (Figure 1). No amino acid sub-

6 1190 CHALLINE ET AL GASTROENTEROLOGY Vol. 135, No. 4 stitutions known to be associated with HBsAg detection failure were found in donors with isolated anti-hbc antibodies. Finally, the brain-dead, heart-beating donor with isolated anti-hbs antibodies (donor 29) had 1 amino acid substitution within the a determinant of HBsAg (Gln to Pro at position 129, Q129P) and 2 substitutions close to it (Gly to Lys at position 112, G112K; and Glu to Gly at position 164, E164G) (Figure 1). Additional amino acid substitutions were observed at various positions in the pres1 pres2-s region in different donors, but they could not be linked to HBsAg detection failure (data not shown). Full-Length prec-c Region Sequence Analysis in the HBV-DNA Positive Donor With Isolated Anti-HBs Antibodies Full-length sequence analysis of the prec-c gene in the HBV-DNA positive organ donor with isolated anti-hbs antibodies showed a precore mutation at position 1896, abolishing HBeAg production. In addition, amino acid substitutions were observed at positions 74, 80, and 155 of the HBc antigen relative to a prototype sequence of the same genotype, 2 of which are located in an immunodominant epitope region spanning residues Discussion We detected HBV DNA in the vast majority of organ, tissue, and cell donors with detectable HBsAg. However, HBV-DNA levels were considerably lower than those reported in patients with chronic hepatitis B, which were of the order of 7.0 and 9.0 log 10 IU/mL on average in recent large-scale clinical trials in HBeAg-negative and HBeAg-positive patients, respectively. 23,24 These low HBV-DNA levels probably reflect the HBV-DNA levels in low-risk, asymptomatic individuals. 25 Low HBV replication levels could explain why HBV DNA could not be detected in several HBsAg-positive patients, some of whom also had anti-hbs antibodies. Although it is not clear whether or not these patients can transmit HBV, they were excluded from donation because of the presence of HBsAg. Two organ donors and 2 cornea donors with isolated anti-hbc antibodies (3.8% and 9.5%, respectively) were found to be HBV-DNA positive. This suggests, in keeping with previous reports, 7 10 that anti-hbc antibodies are a sensitive but poorly specific marker of HBV replication. Although it is reasonable that donors be tested systematically for anti-hbc antibodies and that anti-hbc positive donors be excluded from liver donation, the utility of systematic HBV-DNA testing in such donors is questionable. In the current context of organ and tissue shortage, given the very low likelihood of HBV transmission in the absence of detectable HBV DNA in a sensitive real-time PCR assay, transplantation of organs (other than liver), tissues (other than the cornea), and cells might be considered when anti-hbc antibodies are present but HBV DNA is absent. This approach would be limited to specific groups of recipients and calls for prospective studies. The case of corneas is different because blood from dead donors frequently yields false-positive and false-negative results in serologic tests, 12 as confirmed here. We indeed observed, in a large series of cadaveric cornea donors, a consistently higher proportion of sera positive for at least one viral marker than in the general population and in heart-beating donors, with a significant relationship between the macroscopic aspect of serum at the time of testing and a positive, equivocal, or discrepant result for several markers including serologic HBV markers. 12 The performance, sensitivity, and specificity of nucleic acid testing in such donors remain to be established. There are 2 possible reasons for the lack of HBsAg detection in patients with HBV DNA: (1) low-level, undetectable HBsAg in a patient with low-level replication, and (2) amino acid substitutions in the HBsAg sequence, making it unrecognizable by the antibodies used in usual tests. 21 In organ, tissue, and cell donors with at least 1 serologic marker of HBV infection (excluding isolated anti-hbs antibodies), amino acid substitutions reported to be associated with HBsAg detection failure were found in donors with detectable HBsAg, whereas no such substitutions were found in donors in whom HBsAg was undetectable in 3 different enzyme immunoassays despite ongoing HBV replication. This suggests that HBsAg detection failure was related to very low antigen levels in these donors. The residual risk of HBV transmission among organ donors was only 0.07% in our study (95% confidence interval, 0.01% 0.40%). This underestimates the actual risk of HBV transmission, however, because a single individual may donate to several dozen recipients. We identified one donor with isolated anti-hbs antibodies (vaccination profile) who was repeatedly positive for HBV DNA. In this individual, HBsAg detection failure probably was owing to a combination of previously undescribed amino acid substitutions, including a Gln to Pro substitution at position 129 within the a determinant of HBsAg, and the substitutions Gly to Lys at position 112 and Glu to Gly at position 164, outside the a determinant. This donor may have transmitted HBV to at least one kidney recipient. Only systematic nucleic acid testing could have identified the risk of HBV transmission associated with this donor. The use of anti-hbs antibody testing was questionable in our study. Indeed, its sole utility would be to identify, among donors (excluding cornea donors) with anti-hbc antibodies but without HBsAg, those with a recovery profile who could donate (except for their liver) with no risk of HBV transmission. However, the only organ donor with this type of HBV infection was anti-hbs antibody positive, and nucleic acid test-

7 October 2008 HBV REPLICATION IN GRAFT DONORS 1191 ing provides a better assessment of infectivity, whatever the donor s serologic profile. In summary, our results suggest that too many virus-free organs, tissues, and cells are not used, owing to the presence of HBV serologic markers, whereas a low residual risk of HBV transmission exists in seronegative individuals. Several options can be envisaged to improve both the safety and efficiency of transplantation, especially in the context of severe donor shortages. First, the analytic sensitivity of commercial HBsAg assays should be improved further to detect minute amounts of antigen in individuals with lowlevel replication (this also may apply to the diagnostic setting); second, the ability of commercial assays to detect HBsAg mutants should be improved by including antibodies recognizing all reported mutant sequences and for instance by using antibodies targeting the pre-s region and the second loop of the a determinant (amino acids ); third, the utility and cost-efficacy ratio of systematic HBV-DNA testing should be assessed with the goal of excluding HBV- DNA positive donations not recognized by serologic testing while retaining donations that carry no risk of HBV transmission. Meanwhile, the best means of preventing HBV transmission via organ, tissue, and cell transplantation is vaccination of all candidate recipients. References 1. Alter MJ. Epidemiology and prevention of hepatitis B. Semin Liver Dis 2003;23: Eastlund T. Infectious disease transmission through cell, tissue, and organ transplantation: reducing the risk through donor selection. Cell Transplant 1995;4: Locasciulli A, Alberti A, Bandini G, et al. Allogeneic bone marrow transplantation from HBsAg donors: a multicenter study from the Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Blood 1995;86: Hoft RH, Pflugfelder SC, Forster RK, et al. Clinical evidence for hepatitis B transmission resulting from corneal transplantation. Cornea 1997;16: Natov SN, Pereira BJ. Transmission of viral hepatitis by kidney transplantation: donor evaluation and transplant policies (part 1: hepatitis B virus). Transpl Infect Dis 2002;4: Francisci D, Aversa F, Coricelli V, et al. Prevalence, incidence and clinical outcome of hepatitis B virus and hepatitis C virus hepatitis in patients undergoing allogeneic hematopoietic stem cell transplantation between 2001 and Haematologica 2006;91: Wachs ME, Amend WJ, Ascher NL, et al. The risk of transmission of hepatitis B from HBsAg( ), HBcAb( ), HBIgM( ) organ donors. Transplantation 1995;59: Roche B, Samuel D, Gigou M, et al. De novo and apparent de novo hepatitis B virus infection after liver transplantation. J Hepatol 1997;26: Dickson RC, Everhart JE, Lake JR, et al. Transmission of hepatitis B by transplantation of livers from donors positive for antibody to hepatitis B core antigen. The National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database. Gastroenterology 1997;113: De Feo TM, Poli F, Mozzi F, et al. Risk of transmission of hepatitis B virus from anti-hbc positive cadaveric organ donors: a collaborative study. Transplant Proc 2005;37: Chazouilleres O, Mamish D, Kim M, et al. Occult hepatitis B virus as source of infection in liver transplant recipients. Lancet 1994;343: Challine D, Roudot-Thoraval F, Sabatier P, et al. Serological viral testing of cadaveric cornea donors. Transplantation 2006;82: Zou S, Dodd RY, Stramer SL, et al. Probability of viremia with HBV, HCV, HIV, and HTLV among tissue donors in the United States. N Engl J Med 2004;351: Lefrere JJ, Sellami F, Larderie P, et al. Six years experience testing organ donors for viral markers in France. Transfusion 1997;37: Tugwell BD, Patel PR, Williams IT, et al. Transmission of hepatitis C virus to several organ and tissue recipients from an antibodynegative donor. Ann Intern Med 2005;143: Challine D, Pellegrin B, Bouvier-Alias M, et al. HIV and hepatitis C virus RNA in seronegative organ and tissue donors. Lancet 2004; 364: Stuyver L, De Gendt S, Van Geyt C, et al. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol 2000;81: Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature 1985;317: Vyas GN, Rao KR, Ibrahim AB. Australia antigen (hepatitis B antigen): a conformational antigen dependent on disulfide bonds. Science 1972;178: Carman WF, Van Deursen FJ, Mimms LT, et al. The prevalence of surface antigen variants of hepatitis B virus in Papua New Guinea, South Africa, and Sardinia. Hepatology 1997;26: Weber B. Genetic variability of the S gene of hepatitis B virus: clinical and diagnostic impact. J Clin Virol 2005;32: Tordjeman M, Fontan G, Rabillon V, et al. Characterization of minor and major antigenic regions within the hepatitis B virus nucleocapsid. J Med Virol 1993;41: Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med 2006;354: Lai CL, Shouval D, Lok AS, et al. Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B. N Engl J Med 2006;354: Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006;295: Received March 20, Accepted July 10, Corresponding author: Professor Jean Michel Pawlotsky, MD, PhD, Department of Virology, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, Créteil, France. jean-michel. pawlotsky@hmn.aphp.fr; fax: (33) D.C. and S.C. participated equally in this study. The Cobas TaqMan HBV kits were kindly provided by Roche Molecular Systems (Pleasanton, CA). The authors are grateful to Guillaume Dameron, Françoise Darthuy, Fabienne Dubernet, Patrick Larderie, Jocelyné Rémiré, and Pierrette Rigot for helpful technical assistance. The authors thank Mohamed Jarraya (Banque de Tissus de l Assistance Publique- Hôpitaux de Paris), Patrick Sabatier (Banque Française des Yeux), Jean-Marc Benbunan (Réseau Français de Sang Placentaire), Patrice Guerrini and Jacky Claquin (Agence de la Biomédecine), and all the clinical teams involved in living donor and cell transplantation programs in the hospitals of Asistance Publique-Hôpitaux de Paris for their assistance.