Objective: To assess risk factors for liver-related death, we re-evaluated, after a median follow-up of 25 years, a cohort of 70 Caucasian patients with hepatitis B e antigen (HBeAg) positive chronic hepatitis (CH) at presentation.
Methods: Follow-up studies included clinical and ultrasound examinations, biochemical and virological tests, and cause of death.
Results: Sixty-one (87%) patients underwent spontaneous HBeAg seroconversion. During a median period of 22.8 years after HBeAg seroclearance, 40 (66%) patients became inactive carriers, whereas the remaining 21 (34%) showed alanine aminotransferase elevation: one (1%) had HBeAg reversion, nine (15%) detectable serum HBV DNA but were negative for HBeAg, eight (13%) concurrent virus(es) infection and three (5%) concurrent non-alcoholic fatty liver disease. Liver-related death occurred in 11 (15.7%) patients, caused by hepatocellular carcinoma in five and liver failure in six. The 25-year survival probability was 40% in patients persistently HBeAg positive, 50% in patients with HBeAg negative CH or HBeAg reversion and 95% in inactive carriers. Older age, male sex, cirrhosis at entry and absence of sustained remission predicted liver-related death independently. The adjusted hazard ratios (95% CI) for liver related death were 33 (3.01–363) for persistently HBeAg positive patients and 38.73 (4.65–322) for those with HBeAg negative CH or HBeAg reversion relative to inactive carriers.
Conclusion: Most patients with HBeAg seroconversion became inactive carriers with very good prognosis. The risk of liver-related mortality in Caucasian adults with CH is strongly related with sustained disease activity and ongoing high level of HBV replication independently of HBeAg status.
- hepatitis B e antigen seroconversion, sustained alanine aminotransferase normalisation
- inactive carrier
- HBeAg negative hepatitis
- sustained HBV replication
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- hepatitis B e antigen seroconversion, sustained alanine aminotransferase normalisation
- inactive carrier
- HBeAg negative hepatitis
- sustained HBV replication
Chronic hepatitis B virus (HBV) infection is an established cause of liver-related morbidity and mortality and represents a major global public health problem.1 2 Studies conducted in Asian regions of high HBV endemicity have estimated that up to 40% of individuals with chronic HBV infection will progress to liver cirrhosis, end-stage liver disease or hepatocellular carcinoma (HCC) during their lifetime and that HBV-related liver disease contribute to approximately 20 deaths per 100 000 population each year.3 4 The long-term liver-related morbidity and mortality of chronic hepatitis B in untreated Caucasian patients and predictors of survival deserve further evaluation. In fact, the prognostic value of sustained high levels of HBV replication on survival in untreated Caucasian patients is limited to cohorts of patients with cirrhosis.5–7
We conducted this longitudinal study to elucidate the long-term survival according to virological pattern in a cohort of north Italian patients with chronic hepatitis B who had high levels of HBV replication (HBeAg positive) at diagnosis, during a median period of 25 years.
PATIENTS AND METHODS
A longitudinal study began in Padova, northern Italy, enrolling between 1972 and 1984 seventy consecutive hepatitis B surface antigen (HBsAg) and HBeAg positive patients, 49 male and 21 female with a median age of 29 years and with biopsy proven chronic hepatitis (8 (11.4%) with cirrhosis) at presentation. The clinical, virological and histological details of the study population at entry and the natural history of the disease during a mean follow-up period of 5.0 years (SD 2.3 years) were described previously.8 The follow-up of this cohort of patients has been performed at the Department of Gastroenterology at Verona University Hospital in northern Italy and updated through June 2006.
Follow-up data were obtained during regular clinical controls, on average every 6 months for patients who remained HBeAg positive or seroconverted from HBeAg to anti-HBe, but maintained or developed abnormal ALT levels and on everage every 12 months for inactive HBsAg carriers. By 1986 it became clear that HBeAg seroclearance with ALT normalisation is usually associated with histology improvement,8 thus in the subsequent years follow-up liver biopsies were performed only in patients with active hepatitis. Patients who did not undergo the regular controls were traced through the municipality registries and were phoned and asked to attend again the clinic for evaluation. People who refused to attend the clinic again were interviewed by phone and asked to provide laboratory and liver ultrasound examinations with the consent of their family doctors. Data on patients’ liver-related or other diseases that had been collected through the phone interviews were cross-checked with the archives of their family doctors. The causes of death for those deceased were ascertained by retrieving the death certificates. We were able to trace all patients except two (97.1%) from the initial cohort. The current study was approved by the Verona Hospital Ethics Committee.
Definition of clinical events
The time of observation was calculated from the date of entry into the study until death, liver transplantation or the last observation. The primary outcome was liver related mortality. The occurrence of cirrhosis during follow-up was defined by histology or ultrasonography features suggestive of cirrhosis based on a quantitative scoring system derived from the appearance of the liver margins, parenchymal echotexture, portal vein calibre and spleen diameter,9 10 supplemented with the presence of oesophageal varices or ascites. HCC diagnosis was based on the following criteria: a histopathological examination; a positive lesion detected by at least two different imaging techniques (abdominal ultrasonography, computerised tomography, or angiogram); a positive lesions detected by one imaging technique and α-fetoprotein levels exceeding 400 μg/l. Biochemical remission was defined as the normalisation of serum ALT levels on at least two consecutive determinations obtained at least 3 weeks apart and persisting at last observation. After HBeAg seroclearance with anti-HBe seroconversion, patients were defined as inactive HBsAg carries when ALT values were persistently normal and HBV DNA was persistently negative according to non-polymerase chain reaction (PCR) assays (detection limit, 1.4×105 copies/ml). Active hepatitis, after HBeAg seroclearance, was defined as ALT elevation over twice the upper normal value on at least two consecutive determinations obtained at least 3 weeks apart. HBeAg negative hepatitis was diagnosed when abnormal ALT and HBV DNA seropositivity by non-PCR assays persisted continuously or recurred intermittently after HBeAg seroclearance.
HBsAg, antibody to HBsAg (anti-HBs), HBeAg, antibody to HBeAg (anti-HBe), hepatitis delta virus (HDV) and hepatitis C virus (HCV) antibodies, hepatitis A virus IgM antibodies were detected by conventional serological assays. Serum HBV DNA was first tested by homemade spot hybridisation with a detection limit of 0.1 pg/ml,8 then the Digene Hybrid Capture System assay (Digene, Beltsville, MD) was used, with a detection limit of 0.5 pg/ml (1.4×105 copies/ml). More recently HBV DNA was tested with a commercial PCR assay (Cobas Amplicor HBV Monitor, Roche, Basel, Switzerland) with a detection limit of 200 copies/ml.
Serum HDV RNA was detected by an “in-house” reverse-transcriptase PCR (RT-PCR) in the virology laboratory of the University Hospital of Verona. Briefly, HDV RNA was extracted from 200 μl of serum, reverse transcribed and amplified by one-step RT-PCR in a final volume of 50 μl using a set of primers derived from the carboxyterminal portion of the genomic region coding for the HDV antigen (primers 5′ → 3′, position 886–909 and 1331–1310, respectively). The amplification products were visualised on a 2% agarose gel stained with ethidium bromide. For each test sample a negative and a positive control were included in parallel throughout the entire procedure. Serum HCV RNA was first measured by an “in-house” nested RT-PCR with a detection limit of 100 copies/ml,11 then was assessed using a commercial PCR assay (Cobas Amplicor HCV Monitor, Roche, Basel, Switzerland).
The Mann–Whitney and χ2 and exact tests were used for comparison of continuous and categorical variables between two independent groups. p values lower than 0.05 in two-tailed tests were considered as significant.
The cumulative probabilities of survival were analysed by the Kaplan–Meier method14 and comparison between groups was performed by using the log-rank test.15 Patients who died from causes that were not related to liver disease or were lost to follow-up or responded to antiviral therapy were censored at the time of death, drop out or response to antiviral therapy, respectively. Multivariate analysis was also performed by fitting the Cox proportional hazard models in order to evaluate the role of each prognostic factor when controlling for the others. The assumption of proportionality of hazards functions was assessed using graphical methods as suggested.16
Statistical analyses were performed using the STATA software (Stata Statistical Software release 8.0, 2003; Stata Corporation, College Station, Texas).
Our cohort of 70 HBeAg positive patients was followed for a median period of 25.3 years (range, 1.3–33.1) for a total of 1564 person-years. During this observation period nine (12.8%) patients remained persistently HBeAg positive and 61 (87.2%) underwent spontaneous HBeAg seroclearance with anti-HBe seroconversion (fig. 1). The cumulative probabilities of HBeAg seroconversion were 64%, 93% and 97% at 5, 10 and 25 years of follow-up, respectively. Most patients underwent HBeAg seroconversion during the first 10 years of the study (58 of 61, 95.1%), with an incidence rate of 18.7 per 100 person-years. At enrolment, patients who remained persistently HBeAg positive were older (median age, 51 vs. 27 years) and had a higher prevalence of cirrhosis (44% vs. 6.6%) as compared with those who underwent spontaneous HBeAg seroclearance (table 1). At the time of HBeAg seroconversion, their median age was 30 years (range, 16–65) and 11 (18%) had histological evidence of cirrhosis.
The 61 patients with HBeAg seroclearance were followed for a median period of 22.8 years (range, 0.5–28.4) after HBeAg seroconversion. Forty (66%) out of the 61 patients had sustained ALT normalisation and undetectable HBV DNA by non-PCR tests and were considered inactive carriers; more recently HBV DNA was tested by PCR assay in 15 (37.5%) out of the 40 inactive carriers and was detectable in all cases ranging from 700 to 9800 copies/ml. The remaining 21 (34%) out of the 61 patients experienced active hepatitis; among these 21 patients, ALT elevation was associated with HBeAg reversion in one (1%), detectable serum HBV DNA but HBeAg negative (HBeAg negative hepatitis) in nine (15%), concurrent HCV and/or HDV infection in eight (13%) and concurrent non-alcoholic fatty liver disease (NAFLD) in three (5%) cases (fig. 1). Of the three patients with NAFLD, two had histological features of non-alcoholic steatohepatitis (NASH) and one was obese (BMI 38 kg/m2) with fatty liver diagnosed by ultrasound. All three cases with NAFLD were seronegative for both HBeAg and HBV DNA by non-PCR tests; at the last visit HBV DNA was detectable by PCR ranging from 300 to 8750 copies/ml.
The clinical and biochemical features of the 61 patients with spontaneous HBeAg seroconversion according to clinical and serologic categories during follow-up (40 with sustained remission and 21 with active hepatitis) are given in table 2. The patients who developed HBeAg negative hepatitis or HBeAg reversion had a higher prevalence of cirrhosis at the time of HBeAg seroconversion compared to patients with sustained remission (50% vs. 17.5%, respectively; p = 0.04) (table 2).
Eighteen (45%) of the 40 inactive carriers and one (12.5%) of the eight patients with viral concurrent infections lost their HBsAg and developed the related antibody (table 2). The incidence of HBsAg loss was 2.1 per 100 person-years in inactive carriers.
Among the 18 inactive carriers with HBsAg loss the median interval between HBeAg seroconversion and HBsAg clearance was 13.8 years (range, 1.1–26.9).
A total of 50 patients were alive at the end of the follow-up: among them, 30 (60%) were checked into the hospital, 18 (36%) were interviewed by phone and two were lost at the follow-up. Liver-related death occurred in 11 (15.7%) patients, caused by HCC in five and liver failure in six, and one patient underwent OLT for end-stage liver disease (table 1).
The cumulative probability of survival among all patients was 90% and 86% at 10 and 25 years of follow-up, respectively.
The cumulative probability of survival at 25 years was significantly lower in the nine patients who remained HBeAg positive (40%) and in patients developing HBeAg negative hepatitis or HBeAg reversion (50%) as compared to inactive carriers (95%) (p<0.0001) (fig. 2). None of the patients with concurrent HCV and/or HDV infection or patients with concurrent NAFLD died during the 25 years of observation.
The cumulative probability of survival at 25 years was significantly lower in eight patients with cirrhosis at entry (22.5%) and in 17 patients who developed cirrhosis during follow-up (59%) as compared to 45 patients without cirrhosis (100%) (p<0.0001).
Cox proportional hazard regression model showed that older age at diagnosis, male sex, the presence of cirrhosis at enrolment and the absence of sustained remission during follow-up were significantly associated with an increased risk for liver related death or OLT (table 3). In a subgroup analysis excluding patients with concurrent HCV and/or HDV infection or NAFLD, the risk of liver related mortality or OLT was increased 33-fold in patients with HBeAg persistence and 38-fold in those with HBeAg negative hepatitis or HBeAg reversion as compared to patients with sustained remission (inactive carriers) (table 3).
Ten patients with evidence of high levels of HBV replication (two HBeAg positive and eight with HBeAg negative hepatitis) were still alive in the late 1980s when interferon (IFN) therapy became available and three of them (one HBeAg positive and two HBeAg negative) were treated with IFN. An additional four out of the eight patients with concurrent viral concurrent infections received antiviral therapy. Among the seven treated patients, two (one HBeAg positive and one HBV/HCV co-infected patient) responded to antiviral therapy and were censored from follow-up. The details of IFN schedule and reasons for not treating the remaining patients are provided in the following sections.
Clinical outcome of HBeAg positive patients
Among the nine patients who remained HBeAg positive during a median follow-up period of 6.8 years (range, 1.3–25), eight had histologically documented cirrhosis (four diagnosed at entry and four during follow-up). One patient (with cirrhosis diagnosed during follow-up) responded to lymphoblastoid IFN at the dose of 8 million units thrice weekly for 6 months in the year 1989. The remaining seven patients with cirrhosis died: four for decompensated cirrhosis, one for HCC and two for liver unrelated causes. Among these seven patients, six died in the early 1980s before antiviral therapy became available and one did not receive IFN therapy due to ischaemic heart disease which was the cause of unrelated death in 1999. Only one (1.4%) of the 70 initially HBeAg positive patients is still HBeAg positive and alive without clinical and/or serological evidence of cirrhosis at the last observation in June 2006; the patient had ALT fluctuating from normal to 1.5 times the upper normal value during follow-up and remained untreated.
Clinical outcome of inactive carriers
The clinical outcome of the 40 inactive carriers during a median follow-up period of 23 years (range, 3.2–28.2) after HBeAg seroconversion is shown in fig. 3. Seven patients had histologically documented cirrhosis, three at enrolment and four progressed to cirrhosis during the HBeAg positive phase before HBeAg seroconversion. Of these seven inactive carriers with cirrhosis, two died from HCC 7.7 and 9.4 years after HBeAg seroconversion, respectively, three died of liver-unrelated causes, one is alive and one was lost to follow-up. Of the 33 inactive carriers without cirrhosis, three died of liver unrelated causes and the remaining 30 are alive.
Clinical outcome of patients with active hepatitis after HBeAg seroclearance
The clinical outcome of the 21 patients with HBeAg seroclearance and active hepatitis is illustrated in fig. 4.
One patient with cirrhosis at enrolment showed HBeAg reversion associated with persistent ALT elevation 15 months after HBeAg seroconversion and died of liver failure 3.9 years after sustained HBeAg reversion.
Among the nine patients who progressed to HBeAg negative hepatitis two had histologically documented cirrhosis at the time of HBeAg seroconversion and two developed clinical cirrhosis 3 and 13 years after HBeAg seroconversion, respectively. Among the four patients with cirrhosis, one died of liver failure in the year 1981, one did not respond to lymphoblastoid IFN (8 million units thrice weekly for 6 months) in the year 1987 and underwent OLT 11 years later and the remaining two patients remained untreated because of advanced cirrhosis with low platelet levels at the time IFN therapy became available and died of HCC. All five patients with HBeAg negative hepatitis without cirrhosis are alive. All five patients underwent follow-up liver biopsies after development of HBeAg negative hepatitis showing mild to moderate chronic hepatitis without cirrhosis and none showed liver surface nodularity or other ultrasonographic markers of advanced liver disease at follow-up ultrasound examination. One patient failed to respond to lymphoblastoid IFN (8 million units thrice weekly for 6 months) and the other four remained untreated because of mild histology and no clinical evidence of progressive disease.
Of the eight patients with concurrent viral infections, five developed cirrhosis (histologically confirmed in four cases) during follow-up (one during the HBeAg positive phase and four after HBeAg seroconversion) and three had no clinical, liver ultrasonographic and/or biochemical evidence of cirrhosis at the last observation. Of the five patients with cirrhosis, one HBV/HCV infected patient responded to standard combination treatment with pegylated IFN-α and ribavirin, two anti-HDV/anti-HCV positive (HCV RNA negative) patients failed to respond to recombinant IFNα2a (10 million units thrice weekly for 12 months), one anti-HDV/anti-HCV positive (HDV RNA, HCV RNA and HBV DNA negative by PCR at last observation) patient admitted alcohol abuse and was advised to abstain from alcohol intake and the remaining anti-HDV positive (HDV RNA negative at last observation) patient showed intermittently slightly elevated ALT and remained untreated. Of the three patients without cirrhosis, one anti-HDV positive patient was lost to follow-up, one HBV/HCV infected patient lost HBsAg and refused antiviral therapy for HCV and the remaining HBV/HCV infected patient failed to respond to standard combination therapy with recombinant IFN-α and ribavirin. Excluding one anti-HDV positive patient lost to follow-up and one anti-HCV positive patient who responded to antiviral therapy and censored from follow-up, the remaining six patients are alive and without evidence of hepatic complications in those with cirrhosis.
None of the patients with active hepatitis after HBeAg seroclearance received nucleoside or nucleotide therapy.
The major aim of this longitudinal study was to assess the prognostic significance of sustained high HBV replication levels on the risk of liver-related death in a cohort of 70 Caucasian patients with HBsAg and HBeAg positive chronic hepatitis at diagnosis who were followed for a median period of 25 years with a very low drop-out rate. The number of patients is relatively small, but the study population consists of a well-defined cohort of consecutive patients previously described,8 thus minimising the risk of selection bias. Data on liver-related mortality were accurately obtained in the great majority (97%) of patients. After 25 years, survival was significantly worse in patients who remained HBeAg positive, developed HBeAg negative hepatitis or HBeAg reversion, as compared with inactive carriers (40%, 50% and 95%, respectively). It is of note that the adjusted hazard ratios for liver-related death were 33-fold higher in patients who remained HBeAg positive and 38-fold higher in HBeAg negative hepatitis who had HBV DNA positivity or HBeAg reversion with respect to inactive carriers.
To our knowledge, data regarding the relationship between ongoing HBV replication and liver related mortality in the West are limited to patients who have already progressed to cirrhosis.5–7 Studies conducted in tertiary care centres in Europe among patients with compensated cirrhosis showed that those with persistently high levels of HBV replication, as indicated by HBeAg positivity, have increased liver related death rates, of about 2-fold, as compared to those who clear HBeAg with biochemical remission.5 Early studies on the natural history of chronic hepatitis B in Caucasian patients do not provide information whether HBeAg seroconversion leads to a decreased risk of liver-related adverse events and mortality, due to the too short follow-up period of 5 years at most8 17 18
A prospective population-based cohort study in HBsAg positive Chinese patients has reported that high viral load at baseline (⩾105 copies/ml) is significantly associated with increased mortality from HCC and chronic liver disease over a 11-year period.19 However, these findings should be considered with caution because only baseline measures of HBV DNA level were tested, which are poorly related with the levels of HBV replication and disease activity during the whole follow-up of an individual. Only one prospective study from Asia monitored serum HBV DNA over time and showed that patients with sustained elevation of HBV DNA had the highest HCC risk,20 but data on the risk of liver-related mortality were not reported.
This is the first long-term longitudinal study in untreated adult Caucasian patients with chronic hepatitis B (89% without cirrhosis at diagnosis) showing that ongoing high level of HBV replication during follow-up is associated with significantly increased risk of liver-related mortality. In our study the inactive carrier was defined by sustained normal ALT and HBV DNA persistently negative by non-PCR assays, thus including a heterogeneous group of patients who may have up to 1.4×105 copies/ml of HBV DNA. Nevertheless we showed that HBeAg negative patients with sustained normal ALT and HBV DNA levels of less than 1.4×105 copies/ml have a very good prognosis with long life expectancy. Our data suggest that recent findings from cohort studies in Chinese subjects in their 40s with perinatally or early childhood acquired HBV infection (85% of cohort HBeAg negative, 94% with normal ALT) that serum HBV DNA levels above 104 copies/ml increase the risk for cirrhosis and HCC regardless of serum ALT levels and HBeAg status20 21 can not be directly referred to Caucasian patients. Differences in long-term outcomes between Asian and Caucasian patients should be taken into account in current recommendations for management and treatment of chronic hepatitis B.1 22
Notably, our data are in accordance with a recent longitudinal study of 91 HBeAg positive Italian children showing that 81 (95%) of 85 children without cirrhosis at enrolment and who underwent spontaneous HBeAg seroconversion remained inactive carriers after 29 years follow-up and none died.23 However, of the four children with cirrhosis two remained inactive carriers and two developed HCC 9 and 16 years after HBeAg seroconversion.23 In our study two inactive carriers who had already developed cirrhosis during the HBeAg positive phase, died of HCC 8 and 9 years after HBeAg seroconversion. These findings underline the clinical relevance of HBeAg seroconversion with virological and biochemical remission early in the course of the liver disease before cirrhosis occurrence.
In addition to the main findings on survival, this study provides some data on the long-term rates of serological events. The incidence of HBeAg seroconversion was 18.7 per 100 person-years in the first 10 years of the follow-up and the incidence of HBsAg seroconversion was 2.1 per 100 person-years in accordance with previous studies from Western countries in adults patients.1 2 18 24–26
Our study shows that 34% of patients developed active hepatitis despite HBeAg seroconversion: nine patients (15%) showed HBeAg negative hepatitis and one (1%) showed HBeAg reversion. The patients who developed HBeAg negative hepatitis had a significantly higher prevalence of cirrhosis at the time of HBeAg seroconversion as compared with inactive carriers. These results are in agreement with an Asian study by Hsu et al,27 reporting that 33% of patients with spontaneous HBeAg seroconversion experienced ALT elevation during a median follow-up period of 8.6 years, associated with HBeAg negative hepatitis in 24% of cases and with HBeAg reversion in 4%. In the same study, the patients with liver cirrhosis at the time of HBeAg seroconversion had a 10-fold increased risk of developing HBeAg negative hepatitis. Overall these data support the view that HBeAg negative chronic hepatitis represents a late phase in the natural history of chronic HBV infection.
Concurrent HCV and/or HDV infection was found in a small proportion (13%) of our patients with active hepatitis after HBeAg seroconversion and did not seem to affect the natural history of chronic hepatitis B. The complex viral interplay in cases of dual or triple infection28 may provide an explanation to the clinical evidence in longitudinal studies that HBV/HDV co-infection is not always associated with a more severe disease than HBV infection alone6 and that HBV/HCV concurrent infection is not always associated with a higher risk of HCC than in either infection alone.29
In conclusion, the present long term study in Caucasian patients with chronic hepatitis B shows that most patients become inactive carriers after spontaneous HBeAg seroconversion and this condition confers survival benefit with regard to liver disease mortality, particularly if present before the onset of cirrhosis. HBeAg reversion or progression to HBeAg negative chronic hepatitis occurs in a relatively small proportion of patients following HBeAg seroconversion. The risk of liver-related mortality is strongly associated with sustained high level of HBV replication and disease activity during follow-up, independently of HBeAg status. Older age, male gender and cirrhosis also strongly predict disease-related mortality.
Funding: This study was partially supported by the MURST 60% 2005 of the University of Verona (GF, 2005)
Competing interests: None.
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