Case Report

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The enhanced biotin-streptavidin method (StrAviGen Super Sensitive Alkaline Phosphatase kit, BioGenex Laboratories; San Ramon, California) and highly specific monoclonal antibodies against HAV antigen (BioGenex Laboratories) were used to detect HAV antigen immunohistochemically. The specificity of the reaction was confirmed by analyzing negative control tissues (liver biopsy specimens infected with hepatitis B and C viruses and gallbladders removed because of calculous cholecystitis) and HAV-producing cell lines.

A microscopic examination of the liver showed severe, predominantly lymphocytic portal inflammation, lobular disarray, hepatocyte ballooning, and focal necrosis. Intrahepatic cholestasis and ductular epithelial injury with lymphocytic inflammation and ductular proliferation were present. Immunohistochemical analysis showed faint cytoplasmic staining in approximately one third of the hepatocytes. In contrast, an intense cytoplasmic signal was detected in the epithelial cells of the biliary ducts (Figure 1, top left) and in the ductules and pseudoducts (Figure 1, top left and right).

View larger version (127K): [in this window] [in a new window]   Figure 1. Immunostaining for antigen associated with hepatitis A virus in the liver and gallbladder from a patient with acute hepatitis A. Microscopic examination of the liver showed severe portal inflammation with evidence of pericholangitis and intrahepatic cholestasis, in addition to focal hepatocyte necrosis and ballooning. Immunohistochemical staining of liver showed hepatitis A virus antigen in the biliary epithelium of bile ducts (top left), ductules and pseudoducts (top right), and faint cytoplasmic staining in approximately one third of the hepatocytes. The gallbladder showed a moderate lymphocytic infiltrate with virtual absence of neutrophils. Hepatitis A virus antigen was present in most epithelial cells (bottom left) and was located predominantly in the supranuclear areas of the cytoplasm of epithelial cells (bottom right).

Discussion

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Previous studies in humans have shown HAV antigens and RNA only in hepatocytes and Kupffer cells [5-8]. However, the occurrence of cholecystitis in association with acute HAV infection [2, 3] and the recurrence of hepatitis A after liver transplantation [9] suggest that HAV might infect cells other than hepatocytes. Although HAV replication in cells of extrahepatic origin was documented in various cell lines and in several animal models [4], the presence of extrahepatic HAV infection and, specifically, infection of the biliary epithelium have not been shown previously in humans. We showed that HAV can directly infect the biliary epithelium of the gallbladder and the intrahepatic and extrahepatic bile ducts.

Gallbladder wall thickening is commonly noted during sonographic examination in patients with acute viral hepatitis [10]. Further, acalculous cholecystitis has been reported in children with acute hepatitis A [2, 3]. Our observation indicates that HAV can infect both gallbladder and bile duct epithelium. It is possible that such a direct infection of the biliary epithelium may have a direct role in the pathogenesis of HAV-associated cholangiopathic infection.

Both clinical and experimental data indicate that HAV-induced liver injury is mediated by immunologic mechanisms [4]. In our patient, the inflammatory infiltrate around and within the biliary epithelium consisted almost exclusively of lymphocytes. This finding suggests that cell-mediated immunity may also have a role in the pathogenesis of hepatitis-A-associated cholangiopathies. Further, the presence of HAV within epithelial cells of the biliary tract may provide insights into the mechanisms by which the virus is exported to the bile and excreted with the feces.