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Occult Hep B: Long-term histologic and virologic outcomes of acute self-limited hepatitis B
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Hepatology, May 2003, Volume 37, Number 5. Nobukazu Yuki et al, Department of Gastroenterology, Osaka National Hospital, Osaka, Japan.
"....occult HBV persisted in the livers of all 9 patients studied up to a decade after resolution.... The unresolved problem in the occult HBV infection issue is whether it has any clinical impact.. We must stress that its clinical
relevance remains uncertain... The sample size is not large enough to draw a solid conclusion on factor(s) contributive to liver fibrosis.... the fibrosis stage was related to the viral loads in the acute phase and seemed to be determined by the nature of the disease at the onset.. We cannot completely exclude a possibility that low-level alcohol intake in some patients may have affected their fibrosis stage.... Many studies indicate that occult HBV infection might play a critical role in the development of hepatocellular carcinoma in patients with serologically unidentified pathogenesis of hepatocellular carcinoma..it is conceivable that hepatocellular carcinoma is unlikely to occur in individuals who recovered from acute hepatitis B..
The peak alanine aminotransferase activity in the acute phase ranged from 23.0 to 129.3 times the upper limit of normal (median, 65.3), and the peak bilirubin levels ranged from 1.0 to 35.1 times the upper limit of normal (median, 9.8).... Liver function was normalized within 3 months, and HBsAg was cleared from the serum within 5 months....
.....Liver function data, including serum alanine aminotransferase activity, were normal for all patients. They were all clear of HBsAg, and anti-HBs had developed in 12 patients. All had seroconverted to antibody to hepatitis B e antigen and tested positive for anti-HBc. Using sera diluted 200-fold, anti-HBc was detected in only 1 patient. Overall, all patients showed complete clinical and serologic recovery from acute hepatitis B... Histopathologic examination showed that a low grade of liver inflammation had persisted for a decade."
Abstract: The long-term impact of acute self-limited hepatitis B on the liver is unknown. Fourteen patients were recalled at a median of 4.2 years (range, 1.8-9.5 years) after the onset of acute hepatitis B. All showed clinical and serologic recovery with circulating hepatitis B surface antigen (HBsAg) clearance. Antibody to HBsAg (anti-HBs) had developed in 12 patients. Nine underwent liver biopsies at a median of 7.2 years, and histologic findings were evaluated using Ishak scores. Serum samples and frozen liver tissue were subjected to real-time detection polymerase chain reaction (PCR) to quantify the surface and X regions of the hepatitis B virus (HBV) genome and qualitative PCR to detect the covalently closed circular (ccc) HBV DNA replicative intermediate. Three patients had low levels of circulating HBV DNA up to 8.9 years after the onset, whereas both HBV DNA surface and X regions were found in the liver of all 9 patients examined, including 7 negative for serum HBV DNA. Liver viral loads assessed by the 2 regions showed a significant correlation (r = 0.946; P = .008), and all patients tested positive for ccc HBV DNA. Liver fibrosis and mild inflammation persisted in 8 patients. The fibrosis stage had relation to peak serum HBV DNA in the acute phase (P = .046) but not to liver viral loads in the late convalescent phase. In conclusion, occult HBV infection persists in the liver and is accompanied by abnormal liver histology for a decade after complete clinical recovery from acute self-limited hepatitis B.
Background: The clearance of circulating hepatitis B surface antigen (HBsAg) and appearance of antibody to HBsAg (anti-HBs) with normalization of liver function have been generally accepted as evidence of clinical and serologic recovery from acute hepatitis B. However, in chronic HBsAg carriers, there is growing evidence that hepatitis B virus (HBV) DNA sequences persist in the liver for years after seroclearance of HBsAg and seroconversion to anti-HBs. Although the clinical and pathologic implications of occult HBV infection in the liver are unknown, viral eradication is unlikely to be achieved once chronic HBV infection has been established. The cytotoxic T-lymphocyte (CTL) response is weak or undetectable in chronic HBV infection. In contrast, a vigorous, polyclonal, and HBV-specific CTL response against multiple HBV epitopes is readily detectable during acute self-limited HBV infection. HBV-specific CTLs further persist in the blood for several decades after recovery from acute hepatitis B. In the face of an enhanced immune response leading to disease resolution, the virologic outcomes of acute self-limited hepatitis B may differ from those of HBsAg seroclearance and anti-HBs seroconversion in the course of chronic HBV infection.
At present, the long-term histologic and virologic impact of acute self-limited hepatitis B on the liver is unexplored. Studies using polymerase chain reaction (PCR) to detect HBV DNA sequences have shown that low levels of circulating HBV DNA can persist after clinical and serologic recovery from acute hepatitis B but tend to disappear after long-term follow-up. Peripheral blood mononuclear cells are known as the site of persistent HBV infection long after recovery from acute hepatitis B and may contribute to continuous priming of the HBV-specific CTL response. However, direct evidence is very limited for the possibility of HBV infection persisting in the liver in the late convalescent phase and exerting virulence. To address these issues, liver histologic and virologic outcomes were investigated in patients with a remote history of acute self-limited HBV infection.
Between December 1990 and August 1998, 19 patients with acute hepatitis B were admitted to our institution. A diagnosis of acute hepatitis B was based on
elevated serum alanine aminotransferase activity, detection of HBsAg and immunoglobulin M antibody to hepatitis B core antigen (anti-HBc) in the serum, and the recent onset of jaundice and other typical symptoms. Coinfection with hepatitis A, C, D, or E; Epstein-Barr virus; or cytomegalovirus was ruled out because of the negative results of serologic tests. Confounding etiology of liver disease, human immunodeficiency virus infection, and other immunodeficient diseases were not found in any patient. In 2000, this complete series of patients with acute hepatitis B was contacted for follow-up. Fourteen patients (74%) revisited our institution and were reevaluated for clinical, serologic, histologic, and virologic recovery from the disease. The group comprised 11 men and 3 women ranging in age from 26 to 65 years (median, 43 years). All patients were negative for hepatitis C virus antibody by an enzyme-linked immunosorbent assay (Ortho Diagnostic Systems Co., Ltd., Tokyo, Japan) and serum hepatitis C virus RNA by PCR.14 They had no history of administration of hepatotoxic drugs and showed no evidence of autoimmune liver
disease. Their daily alcohol intake was reevaluated based on a detailed questionnaire and interview. Of the 14 patients, 5 were nondrinkers. Four patients did not have a habit of daily drinking, and their alcohol intake was less than 5 g/d. Alcohol consumption exceeded 5 g/d in the remaining 5 patients, but the levels were relatively low (10-15 g/d in 4 patients and 40-45 g/d in 1 patient). After a median of 4.2 years (range, 1.8-9.5 years) from the onset of the disease, the liver function of the 14 patients was reevaluated using blood chemistry and ultrasonography. Serum samples were obtained from all patients and subjected to serologic and virologic tests for HBV. Ultrasound-guided liver biopsies were performed on 9 patients at 1.8 to 9.5 years (median, 7.2 years) after resolution, and paired serum and liver samples were obtained. The study was approved by the local research ethics committee in accordance with the 1975 Declaration of Helsinki, and all patients provided written informed consent.
RESULTS
Clinical and virologic profiles at the onset of acute hepatitis B
The peak alanine aminotransferase activity in the acute phase ranged from 23.0 to 129.3 times the upper limit of normal (median, 65.3), and the peak bilirubin levels ranged from 1.0 to 35.1 times the upper limit of normal (median, 9.8). HBV genotypes and the peak serum HBV DNA levels were determined for 13 patients. Eleven patients were infected with genotype C, whereas the remaining 2 had genotype A. Real-time detection PCR to quantify the HBV genome was performed on serial serum samples in the acute phase. In each case, the peak point determined by the surface primers coincided with that determined by the X primers. A significant correlation was observed between the peak levels of the HBV DNA surface region (median, 2.0 x 106 ; range, 8.0 x 10(3) to 3.0 x 10(8) copies/mL) and those of the X region (median, 3.6 x 10(5) ; range, 4.7 x 10(3) to 1.1 x 10(8) copies/mL) (r = 0.989; P = .001). Liver function was normalized within 3 months, and HBsAg was cleared from the serum within 5 months.
Clinical and serologic recovery after follow-up
After 1.8 to 9.5 years (median, 4.2 years) from the onset of acute hepatitis B, the 14 patients were examined for clinical and serologic recovery. None of the patients presented symptoms of liver disease, and all were found to have normal livers on ultrasonography. Liver function data, including serum alanine aminotransferase activity, were normal for all patients. They were all clear of HBsAg, and anti-HBs had developed in 12 patients. All had seroconverted to antibody to hepatitis B e antigen and tested positive for anti-HBc. Using sera diluted 200-fold, anti-HBc was detected in only 1 patient. Overall, all patients showed complete clinical and serologic recovery from acute hepatitis B. When sera collected in the late convalescent phase were subjected to HBV DNA PCR, 3 patients (21%) were found to have low levels of circulating HBV DNA up to 8.9 years after the onset of the disease. The HBV DNA surface region of 7.7 x 102 to 2.4 x 104 copies/mL was detected in the 3 patients, whereas the X region of 7.7 x 103 copies/mL was found in only 1 patient. Neither the surface region nor the X region was amplified in the remaining 11 patients.
Liver virologic and histologic outcomes
Virologic and histologic studies were further performed on liver biopsy specimens obtained from 9 patients at 1.8 to 9.5 years (median, 7.2 years) after the onset of the disease. Although only 2 patients remained positive for circulating HBV DNA, both regions of the HBV genome were found in frozen biopsy specimens from all patients by real-time detection PCR using target-specific fluorogenic probes.
Overall, only 1 of the 9 patients examined had normal liver histology. The histologic outcomes were unlikely to be affected by an alcoholic factor. The 9 patients with liver biopsies had low levels of alcohol intake (Table 1). Moreover, similar histologic findings were observed for the 5 patients with very low levels of alcohol intake (nondrinker or <5 g/d, if any). Mild portal inflammation persisted in 4 patients, accompanied by mild focal necrosis in 1 patient. Four patients retained liver fibrosis, and the stage was scored 3 in 2 patients. Mild steatosis was seen in patients 1 and 9, but there was no evidence of pericellular/perisinusoidal fibrosis suggestive of alcoholic or nonalcoholic steatohepatitis.
Liver viral loads and fibrosis stage in the late convalescent phase were further correlated with patient clinical courses. Liver viral loads estimated from the levels of the HBV DNA surface and X regions had no relation to the peak levels of serum alanine aminotransferase activity, bilirubin, and HBV DNA in the acute phase. The relationship was not evident between the liver viral loads and time from the onset of acute hepatitis B. Advanced liver fibrosis stage years after clinical and serologic recovery was associated with high levels of peak serum HBV DNA at the onset (P = .046) but not with the liver viral loads in the late convalescent phase. No relationship was found between liver fibrosis stage and time from the onset of the disease. The influence of alcohol intake on fibrosis stage was also not evident.
Discussion by authors
At present, the long-term impact of acute self-limited hepatitis B on the liver is unknown in humans. The main aims of the present study were to explore the
long-term histologic and virologic outcomes of acute self-limited hepatitis B. The duration and pathologic implications of serologically undetectable HBV
persistence in the liver were investigated. Enhanced HBV-specific immune responses are induced following acute exposure to the virus, which presents a striking contrast to chronic HBV infection and is believed to lead to termination of the disease. However, there is a report of a chimpanzee that continued to harbor a nonreplicating episomal form of HBV DNA in the liver after resolution of acute hepatitis B by PCR criteria. Similar findings of the persistence of woodchuck hepatitis virus (WHV) DNA in the liver have been described for apparently healthy animals after recovery from acute WHV hepatitis.
All patients studied had demonstrated clinical and serologic recovery from acute hepatitis B. Most (80%) of the patients had developed anti-HBs and were clear of HBV from the serum by PCR criteria. Nevertheless, occult HBV persisted in the livers of all 9 patients studied up to a decade after resolution. One report in the literature showed that HBV DNA was detected in the livers of 2 of 4 patients who had had acute self-limited hepatitis B 30 years previously. Collectively, complete HBV eradication seems to be a very rare event, if it occurs at all, after resolution of acute hepatitis B. In the current study, both of the HBV DNA surface and X sequences derived from 2 distinct virus genomic regions were detected in all liver samples tested. A close correlation was further shown between the levels of the HBV DNA surface sequences and those of the X sequences in the liver, which ensured reliable identification of HBV traces. Moreover, all liver samples tested positive by the PCR methodology to detect an intact direct repeat region, which will amplify the ccc HBV DNA but is unlikely to amplify the incomplete virion HBV DNA and integrated HBV DNA. Although we must stress a possibility that some forms of integrated HBV DNA and other HBV replicative intermediates are amplified, the procedure has been used as a fairly specific assay to detect ongoing occult HBV infection. Collectively, these observations suggest that a replication-competent
episomal form of HBV DNA persists in the liver of all patients with a remote history of acute self-limited hepatitis B.
The present study provides direct evidence that liver tissue is a site of virus propagation during convalescence. A previous study showed that HBV infection persisted in the liver but not in peripheral blood mononuclear cells from patients with acute self-limited hepatitis B 30 years earlier, thus indicating that liver tissue can be the
main site of virus propagation in such cases.10 Unfortunately, occult HBV infections in multiple organs were not compared in the present study, which aimed to explore liver histologic and virologic outcomes. We must stress that further studies are obviously necessary to address this issue. The liver HBV DNA levels were not affected by the severity of liver injury and the viral loads in the acute phase. Interestingly, the levels did not decline with the time elapsed after resolution, and low-grade liver inflammation persisted. Taken together, the presence of ccc HBV DNA may imply low levels of ongoing replication. After infected cells have been massively eliminated by virus-specific immune responses, the HBV replication may be balanced against the host immune pressure for years. Vigorous HBV-specific CTL reactivity has been shown in patients years after clinical and serologic recovery from acute hepatitis B. Ample evidence now exists that cytokines (e.g., tumor necrosis factor and interferon gamma) produced by the CTLs play a major role in the posttranscriptional down-regulation of HBV genome expression without significant liver injury. The virus may establish a dynamic equilibrium with the host immune system, where it stimulates the immune response, which in
turn keeps HBV replication at an extremely low level. Further quantitative analysis of the liver ccc HBV DNA will shed more light on this issue.
The unresolved problem in the occult HBV infection issue is whether it has any clinical impact. A persistent replication-competent state of HBV infection in the liver raises clinically important problems in the context of liver transplantation and immunosuppressive treatment. In contrast, in an immune-competent state, there is no clear proof that strongly suppressed HBV infection can be involved in the development of advanced chronic liver disease. In the present study, the liver persistence of the replication-competent virus was accompanied by low-grade liver inflammation. A recent study on woodchucks convalescent from acute WHV hepatitis also showed the lifelong persistence of occult infection in the liver, which induces a very mild liver inflammation continuing for life.21 We must stress that its clinical relevance remains uncertain. Most of our patients retained mild liver fibrosis irrespective of the time elapsed between recovery and liver biopsies. The sample size is
not large enough to draw a solid conclusion on factor(s) contributive to liver fibrosis. However, the fibrosis stage was related to the viral loads in the acute phase and seemed to be determined by the nature of the disease at the onset, although the possibility remains that persistent inflammation exerted some influence on the regression of fibrosis. We cannot completely exclude a possibility that low-level alcohol intake in some patients may have affected their fibrosis stage.
The oncogenic potential of occult HBV after resolution of acute hepatitis B is further doubtful. Many studies indicate that occult HBV infection might play a critical role in the development of hepatocellular carcinoma in patients with serologically unidentified pathogenesis of hepatocellular carcinoma.24,25 Woodchucks that have been infected by the corresponding hepadnavirus (WHV) are at high risk of developing hepatocellular carcinoma after complete resolution of acute WHV hepatitis. In humans, however, long-standing active HBV replication accompanied by significant liver injury is likely to be a very important factor that causes the development of HBV-related hepatocellular carcinoma. Patients with hepatocellular carcinoma with occult HBV may possibly have had chronic HBV infection and lost HBsAg. It is conceivable that hepatocellular carcinoma is unlikely to occur in individuals who recovered from acute hepatitis B.
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