INTRODUCTION

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The spectrum of liver injuries caused by hepatitis B virus (HBV) ranges from self-limited acute infection to chronic hepatitis, cirrhosis, and hepatocarcinoma. Fulminant hepatitis B (FHB) is uncommon, occurring in about 1% of patients with acute hepatitis (26). In the health care setting, HBV infection remains a serious problem. Patient-to-patient transmission is well recognized (2, 13, 16) and is more frequent than either health care worker-to-patient (14, 43) or patient-to-health care worker transmission (12). Infection control measures, including segregation of HBsAg-positive dialysis patients (3) and vaccination (23), have effectively reduced nosocomial HBV acquisition. However, in the hematologic setting specific risk factors, including the high frequency of percutaneous procedures and the presence of intravenous lines, may facilitate the spread of blood-borne pathogens (1). Indeed, several cases of reactivation of HBV infection and/or de novo fulminant hepatitis following withdrawal of immunosuppressive therapy for hematologic and oncologic diseases have been described (9, 22, 30, 37, 45).

The pathogenesis of FHB remains unclear, though both viral and host factors are believed to play an important role (7, 21, 24, 28, 33, 39). In this study, we describe an outbreak of HBV infection that occurred between December 1997 and February 1998 among the patients of a hematology unit (San Salvatore Hospital, Pesaro, Italy), leading to a fatal course in 9 of the 11 cases. We conducted an investigation to identify the source of infection and the route of transmission. Molecular characterization of HBV genomes was conducted through sequence analysis of the entire viral genome isolated from all case patients and the putative source case. We also compared the isolates described in this study with published nucleotide and amino acid sequences from patients with fulminant and nonfulminant type B hepatitis.

	MATERIALS AND METHODS

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Patients. At the end of January 1998, the medical records of all inpatients of the hematology ward from August 1997 to January 1998 were reviewed and the patients were traced to collect information on demographic characteristics, HBV serological status, the presence of a vascular catheter, intravenous and subcutaneous administration of drugs, capillary blood drawing, apheresis, receipt of blood products, bone marrow biopsy and/or transplant, and clinical condition. All the patients who had been present in the ward during the above-mentioned period and were HBV susceptible, of unknown HBV serostatus, or HBsAg positive during hospitalization were asked to give monthly blood samples for HBV serology free of charge for a 6-month period.

Laboratory methods. HBsAg, antibodies to hepatitis B surface antigen (anti-HBs), hepatitis B e antigen (HBeAg), anti-HBe, anti-HBc (immunoglobulin G [IgG] and IgM), and anti-HAV (IgG and IgM) were detected by microparticle enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.). Anti-HCV antibodies were detected by enzyme immunoassay (EIA) (EIA-HCV 3.0, Ortho Diagnostic System, Raritan, N.J.); anti-HDV antibodies (IgG and IgM) and HDVAg were detected by EIA (Sorin Biomedica, Saluggia, Italy); and anti-human immunodeficiency virus types 1 and 2 (HIV-1 and -2) antibodies were detected by EIA capture 3.0 (Sanofi Diagnostics Pasteur, Marnes-Le-Coquette, France). Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) antibodies were also sought by commercially available EIA kits with recombinant antigens (Wampole Laboratories, Dist., Cranbury, N.J.; Diamedix Corp., Miami, Fla.). An amplified solution hybridization assay (Digene hybrid capture system; Digene, Gaithersburg, Md.) was used for semiquantitative detection of HBV DNA in serum.

Other assays. Specific sequences of HCV, hepatitis G virus (HGV/GBV-C), and the newly described transfusion-transmitted virus (TTV) were also sought by reverse transcriptase PCR and hemi-nested PCR techniques, using primers from the 5'-untranslated region of HCV, the NS3/helicase region of HGV, and the putative ORF-1 region of TTV, respectively, as previously described (29, 32, 44).

Statistical methods. Attack rates were calculated for patients exposed and not exposed to potential risk factors, excluding deaths and patients lost to follow-up. The chi square test or, when appropriate, Fisher's exact test was used to evaluate the association of outcome with potential risk factors. The unpaired t test was used to compare group means of nucleotide sequence divergences. Two-tailed P values of <0.05 were considered statistically significant. Calculations were performed using STATA statistical software (release 5.0; Stata Corporation, College Station, Tex.).

Comparative analysis. The sequences of all case patients were compared with published sequences of fulminant and nonfulminant hepatitis B, including those recently reviewed by Sterneck et al. (38) and those described by Stuvyver et al. (40) (accession no. AF090838 to AF090842).

Nucleotide sequence accession numbers. The new sequences described in this paper have been submitted to EMBL and assigned accession no. AJ005084 to AJ005110 and AJ009994 to AJ10007.

	RESULTS

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Acute hepatitis B cases. Eleven cases (four males; mean age, 49.5 years; range, 12 to 68 years) of acute hepatitis B were observed between 9 December 1997 and 20 February 1998, with a 14.5% crude attack rate. In 7 cases, infection likely occurred in late October 1997 (7/11, 63.6% attack rate), and in 4 cases, infection likely occurred in December 1997 (4/10, 40% attack rate). All patients had jaundice; alanine aminotransferase ranged from 207 to about 8,000 U/liter. All tested positive for HBsAg and IgM anti-HBc at admission; HBeAg was transiently detected at low levels (less than 1.5 times the cut-off value) in 4 patients at admission, and it was no longer detectable during hospitalization. HBV DNA levels at admission ranged from 192 to 6,890 pg/ml. Of the 11 HBV-infected patients, 4 had myeloma, 3 had non-Hodgkin's lymphoma, 3 had leukemia, and 1 had Kaposi's sarcoma.

HBV nucleotide sequences. The complete HBV nucleotide genome from 10 case patients (the samples from the 11th patient were not available), from the HR patient, and from 8 HBsAg-positive, acutely infected subjects from the Pesaro area, unrelated to the epidemic, was evaluated. HBV DNA was not detectable in the 2 HBsAg carriers hospitalized between August and December 1997. All samples from the 10 case patients and the HR patient showed high sequence homology. Cloning of the PCR products from the HR patient yielded two closely related sequences for X/pre-C/C/P. Pairwise comparisons were performed between the aligned sequences from case patients and that from the HR patient. The percent divergence ranged from 0.6 to 1.4 against each of the two clones for X/pre-C/C/P (mean, 0.86 ± 0.211) and from 0.1 to 0.2 for pre-S1/pre-S2/P (mean, 0.15 ± 0.052) (Fig. 1). By contrast, the percent divergence between the HR patient and the 8 epidemiologically unrelated patients with acute hepatitis B ranged between 1.9 and 4.1 for X/pre-C/C/P (mean, 3.2 ± 0.905) and between 1.0 and 3.9 for pre-S1/pre-S2/P (mean, 2.83 ± 1.132). The difference between groups was statistically significant (t test for X/pre-C/C/P = 7.154; P = 0.0001; t test for pre-S1/pre-S2/P = 6.705; P = 0.0002), thus confirming the monophyly of the sequences obtained from the 10 case patients and the HR patient. Molecular analysis of the X/pre-C/C/P region was also carried out on serum samples collected from case patients at different time points (2 to 6 weeks) from the onset of clinical symptoms: results show 100% intrapatient sequence homology, evidencing lack of genomic evolution in the short-term follow-up (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Percent nucleotide divergence of aligned HBV sequences between the source patient (HR in the text) and the case patients (1 to 10) and between HR and unrelated patients with self-limited acute hepatitis (11 to 18).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Nucleotide alignments of the sequences spanning the EnII/CP region and the precore ORF from 11 patients with fulminant hepatitis B. Nucleotide substitutions detected within the EnII/CP and the G-to-A substitution at position 1896 of the precore region are indicated.  and  boxes within the core promoter upstream of the regulatory sequence are also indicated. Patients' sequences (p1 to p11) are compared with the reference sequence of HBV, subtype ayw.

Predicted HBV amino acid sequences. Overall, 56 nucleotide mutations predicted amino acid changes in the viral ORFs (Table 3); none of the ORFs was interrupted, except for the precore stop codon mutation detected in all cases. Unique or rare amino acid changes could be predicted in 20 motifs (13 in all cases and 7 only in some clones): 1 in the pre-S/S ORFs (in all cases), 2 in the X ORF (in all cases), 8 in the pre-C/C ORFs (3 in all cases), and 9 in the P ORF (7 in all cases). In the pre-S/S and P ORFs, none of the amino acid substitutions were located in motifs essential to critical secretion or antigenic functions, including the reverse transcription and RNase H domains. Amino acid change at codon 67 of the C ORF was detected in all isolates, whereas codons 63 and 64 were mutated only in some clones; these mutations are located in a motif (residues 60 to 90) in the middle of the core protein, at the tip of surface-exposed, protruding spikes of the viral capsid.

Other virologic assays. All cases tested negative for markers of ongoing infections with HAV, HDV, HCV, CMV, EBV and HGV; TTV DNA sequences were detected in 2 patients, neither of whom died from fulminant hepatitis.

Epidemiologic data. Overall, 159 patients were admitted between August 1997 and January 1998. Forty-one of them either had a past HBV infection  (n, 30) or were immune (n, 11) and were excluded from the analysis. Among those remaining, serologic HBV follow-up could not be performed in 37, either because of death (n, 19) or lack of information (n, 18). Of the remaining 81 patients, 5 were already HBsAg positive before October 1997, but 2 of these could not be traced; the other 76 HBV-susceptible patients underwent serologic follow-up, which ranged from 2 to 6 months (mean, 5.5 months; median, 6 months).

	DISCUSSION

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By the end of March 1998, it was clear that the 11 recent HBV infections in the hematology ward were linked, as shown by an identical pattern of nucleotide substitutions in the HBV sequences of all case patients. The HR patient was the most likely source of infection in the first cluster (October 1997). In the second cluster (December 1997), the likely sources were the HBV-infected patients from the October cluster. Nonetheless, patient-to-patient transmission cannot be ruled out. Based on the review of infection control procedures that evidenced breaks, transmission from sources to cases probably occurred via contaminated vials or supplies shared between patients, as already described in other outbreaks (17, 20, 34). Accordingly, we can hypothesize that contamination occurred during one of the intravascular procedures, e.g., blood drawing and intravascular catheter insertion, management, and heparin flushing, all of which presented the opportunity for blood from the carrier to contaminate staff or equipment (e.g., via contaminated hands, gloves, kidney dish, heparin, or normal saline) and hence the intravenous devices of patients. The detection of an identical pattern of nucleotide substitutions in the sequences from the HR patient and the infected patients confirms that a mutated virus was efficiently transmitted to the hospitalized patients.

In previous studies, HBV strains with point mutations in the precore region and/or in the basic core promoter have been detected in patients with FHB (6, 15, 18, 24, 28, 36). Specifically, the combination of the HBV variant with a stop at codon 28 of the precore and two mutations in the core promoter (A-to-T at position 1762 and G-to-A at position 1764), together with several point mutations in all regions of the genomes (5, 38, 40), has been reported in European patients with a severe course of acute HBV infection. However, the association between precore defective HBV strains and the development of acute liver failure has not been documented in all studies (10, 24, 25, 27) or has been revealed only during the late course of acute infection (35). Gunther et al. (11) recently described a novel class of HBV variants bearing mutations in the EnII/CP region which resulted in defective HBeAg expression and enhanced replication in immunosuppressed patients with severe liver disease. This particular phenotype depended mainly on the presence of deletions, insertions, and duplications in the EnII/CP region and, to a lesser extent, on single nucleotide changes: in particular, mutations at positions 1762 and 1764 were not associated with altered expression of viral proteins or with increased levels of viral replication, as also observed by Takahashi et al. (41) in chronic hepatitis patients. More recently, Sterneck et al. (39) suggested that viral variants with enhanced replication competence and/or a defect in HBeAg expression contribute to the pathogenesis of some FHB cases, though additional viral or host factors should also be considered in most cases.

One remarkable feature of this outbreak is the high case fatality rate (81.8%), which is unusual for acute HBV infection (26), although a similarly high rate has been observed in other nosocomial HBV outbreaks (17, 28, 42). The presence of the G-to-A mutation at nucleotide 1896 accounts for the anti-HBe status of the HR patient and for the absence or transient presence of circulating HBeAg in the newly infected patients. The presence of weak though detectable reactivity for HBeAg in some cases is not unexpected (28). Indeed, the presence of HBeAg in the serum of cases with fulminant and chronic HBV infection with no detectable wild-type precore sequences has been previously reported (21, 36, 38). However, cloning of amplified DNA from the HR patient and the case patients did not yield a minor population of wild-type virus expressing the HBe protein. The possible role of mutations in the core promoter sequence of the HBV genome in the pathogenesis of fulminant hepatitis is a controversial issue. In our study, we detected point mutations in the core promoter and in the precore regions of all viral isolates, leading to altered expression of HBe protein. However, the viral replication levels detected in vivo by hybridization and PCR assays were similar to those revealed in unrelated cases of self-limiting acute hepatitis B (data not shown). Stuyver et al. (40) recently reported on three cases of subfulminant hepatitis B among heart-transplanted patients: they suggest that additional amino acid changes located in residues 60 to 90 of the C ORF (a motif containing much of the HBcAg-related antigenicity) could affect the antigenic properties of the core protein. We detected a mutation at codon 67 (T-to-N/S) in all isolates from case patients and the source patient, together with a few other amino acid changes randomly distributed within the C ORF, which would not affect the putative capability of viral particles to present modified T-cell epitopes. Since mutations in the core region were previously described in cases with an aggressive course of acute hepatitis B (8), we believe that further experimental evidence is needed of the putative role of these mutations in the pathogenesis of FHB.

Indeed, the lack of induction of a tolerance phase in the absence of HBeAg secretion (leading to an accumulation of intrahepatic viral particles harboring mutated HBcAg, which are massively attacked by cytotoxic T lymphocytes) is contradicted by the evidence of FHB cases infected with wild-type precore viruses (10, 24, 27). In the viral isolates from all cases, we also detected nucleotide substitutions (and predicted amino acid changes) randomly distributed within the other viral ORFs. However, none of these mutations was located within structurally and antigenically essential motifs. These data confirm previous observations indicating that neither severity nor outcome of infection is influenced by any additional mutation in the HBV genome (19). Since it is conceivable that HBV is not directly cytopathic, other nonviral factors may be responsible for an aggressive course of infection. These host factors may include altered immune response to specific viral epitopes, the HLA environment of hosts where immunosuppressive therapy is being administered or has been withdrawn, and finally, the underlying severe disease affecting the HBV-infected patients. While further study of the particular HBV-host interplay in patients with hematologic disorders is clearly necessary, the present data do not support the hypothesis that viral factors alone were involved in the tragic clinical outcome of these cases.

This is one of the largest hospital-acquired hepatitis B outbreaks in the literature, with an extremely high case fatality rate. However, had the case fatality rate been lower, the spread of HBV among patients might have gone unnoticed. Similar episodes with wild viruses may occur more often than is currently recognized, leading to chronic disease and delayed mortality. The findings of this study strongly warrant efforts to increase personnel awareness of blood-borne infections in hematology facilities, to ensure that standard precautions are taken and to restrict the use of multidose vials. Moreover, patients receiving treatment for hematologic and oncologic diseases should be given a vaccination against HBV, which in Italy is free to all immunosuppressed patients (31).