—  SPECIALTY CONFERENCE  —

Liver Pathology

Case 3 - Chronic Hepatitis, HBV and Co-existent Chronic Schistosomiasis

Dina Tiniakos
University of Athens
Athens, Greece





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Clinical Summary
The patient, a lean 39-year-old woman from the Philippines, was found to be HBsAg positive 14 years prior when she immigrated to Greece. There was no other significant past medical history. In the spring of 2005, during evaluation for employment, there was an abdominal ultrasound examination which showed signs of chronic liver disease and focal fatty change. At that time liver tests were normal. Viral hepatitis panel showed: HBsAg +, HBeAg -, anti-HBe +, anti-HBc +, anti-HBs -, anti-HAV IgG +, ΗCV and ΗDV markers -. Her HBV DNA was 131,500 copies/ml.

Six months later, the patient presented with acute exacerbation of chronic viral hepatitis with AST 2399 IU/L, ALT 2682 IU/L and γ -GT 251 IU/L. One month later when liver tests had rapidly begun to resolve, a percutaneous liver biopsy was performed. At the time of the biopsy the liver tests were: AST 61 IU/L, ALT 129 IU/L, alkaline phosphatase 74 U/L , γ-GT 211 U/L, total bilirubin 0.8 mg/dl, direct bilirubin 0.5 mg/dl and albumin 4.0 mg/dl. There was no change in her viral hepatitis markers. The liver biopsy is submitted for review.


Case 3 - Slide 1
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Case 3 - Figure 1 - HE Lo - Low power view of liver biopsy (H&E)
Case 3 - Figure 2 - Portal and lobular inflammation (10x, H&E)
Case 3 - Figure 3 - Zone 3 inflammation and necrosis (10x, H&E)

Case 3 - Figure 4 - Fibrosed portal tract (10x, H&E)
Case 3 - Figure 5 - Higher power view of Figure 4 (20x, H&E)
Case 3 - Figure 6 - Granulomatous inflammation with multinucleated giant cells (10x, H&E)

Case 3 - Figure 7 - Hepatocytes with ground-glass cytoplasmic inclusions (40x, H&E)
Case 3 - Figure 8 - Trichrome lo - Low power view of Masson Trichrome stain


Histological Features
The biopsy consisted of two thin cores of liver tissue with 20 portal tracts. Most portal tracts showed marked fibrous expansion and a moderate mixed inflammatory cell infiltrate, composed of lymphocytes, macrophages, some plasma cells, few neutrophils and rare eosinophils, extending focally to the periportal parenchyma (mild interface hepatitis). In three portal tracts, epithelioid, non-caseating granulomas with multinucleated giant cells and centrally located, oval-shaped, calcified material were seen. In other portal tracts, the calcified material was surrounded by fibrous tissue and tortuous arteries with thickened and/or hyalinized wall and numerous thin-walled vessels were present.

Liver parenchyma showed scattered necroinflammatory foci, confluent necrosis in acinar zone 3 and rare apoptotic bodies. In many hepatocytes the central part of the cytoplasm was finely granular with a characteristic "ground-glass" appearance and a clear halo. Pale, "sanded", nuclei were noted in some hepatocytes. Rare ovoid, calcified material was present in zone 3.

Masson trichrome stain highlighted portal fibrosis with portal-portal and occasional portal-central fibrous septa without significant architectural distortion. The calcified ovoid material was Periodic Acid Schiff (PAS)-positive, diastase resistant, and on high magnification a distinct thin, pale, outer shell was identified. Ziehl-Neelsen stain was negative and reticulin stain revealed centrally-located dark granules. The ground-glass cytoplasmic inclusions in hepatocytes were PAS-negative and orcein-positive. Immunohistochemistry showed that they were also strongly positive for HBsAg, while mild cytoplasmic and membranous positivity for HBsAg was noted in other hepatocytes as well. Nuclear and mild cytoplasmic immunoreactivity for HBcAg was present in some hepatocytes.

Based on the liver biopsy findings and the clinical history, chronic hepatitis B with moderate activity and bridging fibrosis was diagnosed.The ovoid calcifications with the distinct outer shell which were identified as calcified parasite eggs, the granulomatous inflammation and excessive portal fibrosis were attributed to chronic schistosomiasis, a parasitic zoonosis endemic in the native country of the patient. One month after the biopsy the patient started treatment with pegylated interferon- α2a. Six months later her HBV-DNA had dropped to 1,880 copies/ml while AST and ALT were only mildly elevated. Fecal examination was negative for schistosomal eggs and serological assays were negative for schistosoma-specific antibodies. The patient recalled that many years ago, while in the Philippines, she may have been treated for schistosomiasis. Ultrasound examination did not reveal splenomegaly and the patient did not have symptoms or signs of portal hypertension.

Final Diagnosis
Chronic hepatitis, HBV and co-existent chronic schistosomiasis.

Discussion
Schistosomiasis (bilharzia) is a tropical parasitic disease cause by trematode flatworms of the genus Schistosoma infecting 200 million people worldwide [1]. Hepatic or hepatosplenic schistosomiasis is caused mainly by S. mansoni (Africa, Arabian peninsula, South America), S. japonicum (China, Philippines, Indonesia, Thailand) and S. mekongi (Laos, Cambodia). Hepatic schistosomiasis is characterized by two distinct syndromes: an early inflammatory and a late fibrotic liver disease. The early inflammatory liver disease is the result of a hypersensitivity reaction to live eggs trapped in portal vein branches. Histologically, consists of an eosinophil-rich inflammatory cell infiltrate or even eosinophil abcess surrounding the egg. In chronic disease, epithelioid granulomas with or without multinucleated giant cells are formed. The eggs gradually degenerate and in long-lasting lesions empty, sometimes calcified, shells maybe seen. The late fibrotic stage of hepatic schistosomiasis is characterized by typical extensive portal fibrous expansion, the so called "clay-pipe stem fibrosis", first described by Symmers in 1904 [2]. The progressive occlusion of portal veins eventually results in pre-sinusoidal portal hypertension. Indeed, chronic hepatic schistosomiasis is the most common cause of portal hypertension in the world and usually develops in young and middle-aged adults with long-standing infection and some immunogenetic predisposition [3].

The microscopic examination of excreta for eggs is the gold standard for the diagnosis of schistosomiasis [1]. However, light infection, even with concentration methods, may be missed. Antibody-based serological assays are quite sensitive but cannot distinguish history of exposure from active infection. They give positive results for at least 2 years after cure and some-times longer [1].

Exact identification of the pathogenic schistosoma species based on the evaluation of the eggs in the liver biopsy is difficult. S. mansoni eggs are 60x140 μm in size, have a prominent lateral spine and do not calcify [4]. S. mekongi eggs measure 50x60 μm and have a small lateral knob. Both have an acid-fast shell by Ziel-Neelsen stain. S. japonicum eggs measure 60x85 μm, have a small not easily identifiable lateral spine, their shell may not be acid-fast and may calcify [1, 5]. In the present case, patient's origin from the Philippines, where S. japonicum is the only schistosoma species found [6] and egg characteristics (size, calcification) point to S. japonicum as the most possible cause for the granulomatous liver disease and the excessive portal fibrosis. Infection with Capillaria hepatica, a nematode which rarely may be responsible for human liver disease producing granulomatous hepatitis, should be considered in the differential diagnosis because the eggs are similar to those of schistosomas. However, they have characteristic striated shells and bipolar plugs [7] which were not seen in this case.

Other sources of calcified material in a liver biopsy for non-neoplastic disease include amorphous calcifications within granulomas of infectious origin, such as in tuberculosis or brucellosis [8]. Severe hypercalcemia in hyperparathyroidism may be the cause of calcification in areas of hepatocellular necrosis [9]. Diffuse hepatic calcification has been reported during hemodialysis [10] and dystrophic calcification may rarely develop in ischemic hepatitis [11].

Chronic hepatitis B is the leading cause of chronic liver disease world-wide with 400 million people infected by hepatitis B virus (HBV). The disease is highly prevalent (>10%) in China, Southeast Asia and sub-Saharan Africa [12]. In the present case, immunohistochemistry for HBV antigens was consistent with high viral load and replicative activity. The ground-glass hepatocytes observed in this liver biopsy are a hallmark of chronic HBV infection. They are cells containing large amounts of HBsAg in their proliferated smooth endoplasmic reticulum, producing a fine, granular eosinophilic cytoplasmic inclusion, usually with a clear halo [13]. Ground-glass cells are PAS-negative, are high-lighted with Shikata's orcein and Victoria Blue histochemical stains and are positive by HBsAg specific immunostaining [14]. Their differential diagnosis on an H&E stained section, includes oncocytic change of hepatocytes in chronic liver disease presenting with granular, densely eosinophilic cytoplasm due to large numbers of mitochondria [14]. Ground-glass cytoplasmic inclusions may also be identified in Lafora's disease, cyanamide therapy, glycogenosis type IV and fibrinogen storage disease [14, 15, 16]. Recently, similar inclusions have been shown to occur in individuals without chronic HBV infection or any of the above mentioned well known etiologies. They are thought to be the result of disturbed glycogen metabolism possibly due to multiple therapeutic agents and most of them represent inclusions of abnormal glycogen [15, 16].

Chronic HBV infection is common in areas where schistosomiasis is endemic, thus co-infection with both infective agents is not uncommon. Chronic hepatitis B is more severe and cirrhosis is more likely to develop in schistosomiasis patients [17, 18]. The differential diagnosis of chronic hepatic schistosomiasis with severe portal fibrosis from cirrhosis due to chronic viral hepatitis may be difficult in needle liver biopsies of patients from endemic areas. In liver biopsies with hepatic schistosomiasis, there is usually granulomatous inflammation restricted to the portal and periportal areas, interface hepatitis is uncommon, parenchymal necro-inflammatory changes are absent and acinar hepatic architecture is preserved. In contrast to chronic viral hepatitis with cirrhosis, hepatocellular function and indices remain largely unaffected in chronic schistosomiasis with severe fibrosis [3]. Mild schistosomal-related fibrosis may resolve after successful anti-elminthic therapy [1].

References
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  2. Symmers WStC. Note on a new form of liver cirrhosis due to the presence of the ova of Bilharzia haematobia. J Pathol Bacteriol 1904; 9:237-9.

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  7. Choe G, Lee HS, Seo JK, Chai JY, Lee SH, Eom KS et al. Hepatic capillariasis: first case report in the Republic of Korea . Am J Trop Med Hyg 1993; 48:610-25.

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  10. Sugiura H, Yoshida K, Nakanuma Y, Ohta G, Izumino K, Takazakura H. Hepatic calcification in the course of hemodialysis. Am J Gastroenterol 1987; 82:786-9.

  11. Shibuya A, Unuma T, Sugimoto T, Yamakado M, Tagawa H, Tagawa K et al. Diffuse hepatic calcification as a sequela to shock liver. Gastroenterology 1985; 89:196-201.

  12. Lau GKK. Does treatment with interferon-based therapy improve the natural history of chronic hepatitis B infection? J Hepatol 2007; 46:6-8.

  13. Hadziyannis S, Gerber MA, Vissoulis C, Popper H. Cytoplasmic hepatitis B antigen in «ground-glass» hepatocytes of carriers. Arch Pathol 1973; 96:327-30.

  14. Theise ND, Bodenheimer HC, Ferrell L. Acute and chronic viral hepatitis. In: Burt AD, Portmann B, Ferrell LD, eds. MacSween's Pathology of the liver. 5th ed. London: Churchill Livingstone; 2007:399-441.

  15. Lefkowitch JH, Lobrito SJ, Brown RS, Emond JC, Schilsky ML, Rosenthal LA et al. Ground glass, polyglucosan-like hepatocellular inclusions: A "new"diagnostic entity. Gastroenterol 2006; 131:713-8.

  16. Wisell J, Boitnott J, Haas M, Anders RA, Hart J, Lewis JT et al. Glycogen pseudoground glass change in hepatocytes. Am J Surg Pathol 2006; 30:1085-90.

  17. Aquino RT, Cheffi PP, Catunda SM, Araujo MF, Ribeiro MC, Taddeo EF et al. Hepatitis B and C virus markers among patients with hepatosplenic mansonic schistosomiasis. Rev Inst Med Trop Sao Paulo 2000; 42:313-20.

  18. Conciecao MJ, Argento CA, Chagas VL, Takiya CM, Moura DC, Silva SC. Prognosis of schistosomiasis mansoni patients infected with hepatitis B virus. Mem Inst Oswaldo Cruz 1998; 93 (suppl I):255-8.