Journal of Gastroenterology and Hepatology
May 2005

MATHIAS LICHTERFELD* , SUSANNE HAAS + , HANS-PETER FISCHER + , ESTER VOIGT*, JÜRGEN K ROCKSTROH* AND ULRICH SPENGLER* Departments of *General Internal Medicine and + Pathology, Universitätsklinikum Bonn, Germany and Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

"……These study findings contrast with the results of two other studies in HIV-HCV co-infected intravenous drug abusers, which suggested the extent of hepatic fibrosis to be directly correlated to the level of CD4+ T-cell depletion. Although our small study cannot exclude such an association, it shows that HCV infection can lead to severely impaired liver function even when classical histological lesions of advanced chronic liver disease are not present……"

Background: Liver failure is an increasing cause of death in human immunodeficiency virus-hepatitis C virus (HIV-HCV) co-infected patients. Here, histopathological features of fatal liver disease in HIV-HCV co-infected patients were comparatively assessed.

Histopathology: The science concerned with the cytologic and histologic structure of abnormal or diseases tissue.

Methods: Liver biopsies of seven HIV-HCV co-infected patients with clinically imminent liver death and advanced immune deficiency were studied. Biopsies of seven asymptomatic patients with stable hepatic and immune functions, who were matched according to their documented duration of HIV-HCV co-infection, served as controls. Inflammatory and fibrotic changes as well as hepatocellular steatosis and cholestasis were assessed semiquantitatively by established scores.

Results: All patients with fatal liver disease had severe immunodeficiency and jaundice, while biliary ducts were patent. Unexpectedly, the extent of hepatic steatosis, inflammatory activity and fibrosis was strikingly similar in both study groups. Importantly, liver failure was observed even in the absence of marked fibrosis. Lobular bilirubinostasis was the only feature that significantly distinguished patients with advanced immunodeficiency and fatal liver disease from the control group.

Conclusion: Thus, rapid deterioration of liver function and death can occur in HIV-HCV co-infected patients with advanced immunodeficiency even when liver histology does not reveal markers of end-stage liver disease. Jaundice and marked bilirubinostasis in the absence of biliary tract obstruction seem to herald this complication of chronic hepatitis C in HIV infection.

Due to shared routes of viral transmission, co-infection with the human immunodeficiency virus (HIV) and the hepatitis C virus (HCV) is frequently encountered in certain risk groups such as hemophiliacs or individuals with intravenous drug abuse. Meanwhile it has been well recognized that HIV-HCV co-infected patients suffer from accelerated HCV disease progression, with end-stage liver disease being attained considerably more rapidly than in HCV mono-infected patients. 1,2 On average HCV viral loads are also markedly increased in patients with concomitant HIV infection. 3,4 This more aggressive course of HCV-related liver disease in HIV-HCV co-infected patients seems to be linked to the extent of HIV-mediated cellular immune deficiency. 2 Thus, chronic hepatitis C has been considered as an opportunistic disease of HIV-infected patients. 5

Highly active antiretroviral combination therapy (HAART) can effectively suppress HIV replication, leading to immune recovery as well as a resolution of opportunistic infections. Although HAART does not induce reduction of HCV replication in HIV-HCV co-infected patients, even when profound immunological recovery is achieved, 6,7 its administration seemed to reduce or delay the progression of chronic hepatitis C to end-stage liver disease. 8 Moreover, spontaneous clearance of HCV has also occasionally been observed following the initiation of HAART. 9 Thus, there is emerging evidence that preservation of immune function due to HAART may exert beneficial effects on the clinical course and outcome of HCV-related liver disease in HIV-HCV co-infected patients.

Histopathological findings in liver biopsies from HIV-HCV co-infected patients have been reported only infrequently. 10,11 Thus, the relationships between the degree of cellular immune deficiency and hepatic histopathological findings have not been well established in HIV-HCV co-infected patients. In particular, it is not clear whether imminent liver-related death correlates to specific histological findings in this patient group. To address this issue, we studied liver specimens of seven patients with clinically severe liver disease, all of whom had died from HCV-related liver disease within 6 months after the liver biopsy had been taken. Liver biopsies of seven asymptomatic patients with stable liver disease, who had a similar known duration of HCV and HIV infection, served as a control group.

Here, we studied histopathological features in liver biopsies of HIV-HCV co-infected patients with severely impaired hepatic function, who died from liver failure shortly after the biopsies were obtained. The HIV-HCV co-infected patients with a similar duration of documented viral infections but stable hepatic function served as a reference group. Unexpectedly, the overall degree of hepatic fibrosis as assessed by the Ishak score was almost identical in both groups, despite the fact that all patients in the first group suffered from hepatic failure and deteriorating cellular immune deficiency, while the patients of the control group had only asymptomatic liver disease and stable immune function. Moreover, necroinflammatory changes were similarly pronouced in both patients groups despite their divergent immunological and clinical characteristics. In addition, macrovesicular hepatic steatosis was a common finding in many of our patients, but was neither correlated to CD4+ T-cell counts, nor to the clinical degree of hepatic organ dysfunction nor to previous drug exposure. Microvesicular hepatocellular fat accumulation or steatohepatitis, which occurs occasionally in patients exposed to antiretroviral therapy with nucleoside analog reverse transcriptase inhibitors, 14 was not detected in any of our patients. Thus, our study does not reveal a relationship between the degree of HIV-mediated cellular immune deficiency and the presence of histologically advanced liver disease in HIV-HCV co-infected patients. This finding contrasts with the results of two other studies in HIV-HCV co-infected intravenous drug abusers, 15,16 which suggested the extent of hepatic fibrosis to be directly correlated to the level of CD4+ T-cell depletion. Although our small study cannot exclude such an association, it shows that HCV infection can lead to severely impaired liver function even when classical histological lesions of advanced chronic liver disease are not present. Importantly, other reasons of fatal liver diseases, such as chronic alcohol abuse, certain genetic or metabolic disorders or HIV-associated opportunistic infections were clearly not present in our study individuals and therefore cannot account for these findings.

Canalicular and hepatocellular bilirubinostasis was the only conspicuous histopathological feature in our study, which significantly distinguished between the patients with stable or severely compromised hepatic function. This histological observation corresponded closely to the fact that increased markers of cholestasis were the leading biochemical abnormality in the clinical presentation of patients with fatal hepatitis C. In general, bilirubinostasis associated with minimal inflammatory activity is rarely encountered in patients with acute or chronic viral hepatitis, but can be associated with large bile duct obstruction, certain drug toxicities or sepsis. 17 Yet, extrahepatic bile flow obstruction was ruled out in all patients in the present study by ultrasound or cholangiography and none of our patients was suffering from sepsis or had been exposed to drugs that are known to cause cholestatic liver injury. Moreover, our histological analysis could also exclude steatohepatitis as a further possible cause of cholestatic liver injury. Thus, at present the precise cause for this type of parenchymal cholestasis remains unclear in our patients. Nevertheless, it is quite likely that bilirubinostasis reflects HCV infection under the conditions of severely advanced cellular immune deficiency that was present in all our patients with impaired liver function. Of note, cholestatic liver injury has also been observed in a variety of other immunocompromised HCV-infected hosts such as patients with primary hereditary hypogammaglobulinemia, 18 or organ recipients treated with high-dose immunosuppressive therapy for recurrent rejection. 19,20 In these patients, a broad range of histological changes has been observed ranging from marked bilirubinostasis with little inflammatory or fibrotic changes to severe fibrocholestatic hepatitis. Fibrocholestatic hepatitis C has also been reported in two HIV-positive patients with severe cellular immune deficiency, 21 and in an HIV-HCV co-infected hemophiliac who developed recurrent hepatitis C after liver transplantation. 22 In contrast to these reports, the typical histopathological features of fibrocholestatic hepatitis were clearly not present in our patients. Importantly, in the present study, bilirubinostasis was not necessarily associated with prominent fibrosis, and necroinflammatory changes could be mild to moderate. This observation could reflect a direct cytopathic effect of HCV that might have contributed to the cholestatic liver injury in our immunosuppressed patients. Indeed, extremely high levels of HCV loads in the peripheral blood have been reported for some patients with cholestatic liver injury and fatal hepatitis C. 23,24 Because HCV viral loads had not been determined in the present patients at the time of liver biopsy, we are unable to draw further conclusions on the pathophysiological role of excessive HCV replication for the development of fatal liver disease in our patients.

We cannot exclude with certainty that the liver disease in our patients may have progressed rapidly to fibrocholestatic hepatitis C or cirrhosis after the biopsy, because autopsy specimens were not available in any of our study subjects. However, this possibility seems to be quite unlikely considering the very short survival time of the patients (such as 3 weeks in patient 3), and the fact that the pattern of biochemical abnormalities did not change until death. Moreover, severe clinical liver disease was already present at the time of liver biopsy.

In summary, we observed a broad spectrum of histological findings in seven HIV-HCV co-infected patients with clinically severe liver disease. Intrahepatic cholestasis, however, was the only histological feature that distinguished biopsies of patients with a short-term fatal outcome from those of HIV-HCV co-infected patients with stable asymptomatic liver disease of the same duration, while histopathological scores of inflammation, necrosis, fibrosis and metabolic changes were equivalent between the two groups. Therefore, hepatic bilirubinostasis may herald rapid deterioration of liver function and a fatal outcome in HIV-HCV co-infected patients, even when additional histopathological signs of end-stage liver disease are missing.

All seven patients with fatal HCV-related liver disease were jaundiced, and the presence of marked fatigue (n = 3), hepatic encephalopathy (n = 2) or ascites (n = 3) and reduced synthesis of clotting factors (n = 7) also indicated severely impaired liver function. Cumulative known duration of HCV and HIV infection was >15 and 10 years, respectively. All patients had advanced HIV-associated immunodeficiency with CD4 counts below 60 cells/µL and had experienced at least one extrahepatic manifestation of AIDS (Table 1). With the exception of patients 1 and 7, who had taken azidothymidine monotherapy for more than 1 year, none of our patients had received antiretroviral drugs or other potentially hepatotoxic medication. The patients died from liver failure between 3 and 20 weeks after the biopsies. None of them had clinical evidence of an acute infection or sepsis.

Table 1 Study patient characteristics

Control group (patients with stable immune function)

In contrast to this severely immunocompromised group, all patients in the reference group were asymptomatic. In two patients of the control group (8 and 9), portal hypertension could be inferred by detection of esophageal varices (stage I) and moderate splenomegaly. None of the other patients had signs of advanced liver disease. All patients in the control group had stable CD4 counts and clinically a well-preserved immunological state (Table 1). In six of these individuals, stable HIV infection was maintained by HAART since 1996, while the remaining patient achieved stable HIV disease spontaneously without any antiretroviral therapy. The known cumulative duration of HCV and HIV infection in the control group was likewise >15 and 10 years, respectively.

Biochemical findings and histopathological results of the two patient groups are summarized in Table 2. The liver specimens were compatible with chronic hepatitis in all 14 patients but showed a broad spectrum of fibrosis ranging from slight portal fibrosis to complete cirrhosis in each group. None of the patients had histological evidence of opportunistic liver disease associated with HIV infection or features of drug-induced liver injury. Although both patient groups clearly differed with regard to their clinical presentation and immunological state, analysis of the liver biopsies revealed a similar degree of hepatic inflammation, portal and lobular fibrosis as well as hepatic iron deposition in the two groups. Hepatic steatosis tended to be slightly more pronounced in the patients with severe clinical liver disease (mean proportion of hepatocytes with intracellular fat accumulation: 5.4% vs 3.6%), yet this difference was statistically not significant (P = 0.27, Mann-Whitney U-test). The only differential finding between our two groups was the presence of marked canalicular and hepatocellular bilirubinostasis in the patients with clinically severe liver disease corresponding to their high levels of serum bilirubin and alkaline phosphatase. The difference in the cholestasis score (mean score 1.28 vs 0.14) was statistically significant (P = 0.02, Mann-Whitney U-test), although the number of available samples was rather small for statistical evaluation. Importantly, in some of our patients with severe liver disease, pronounced canalicular and hepatocellular cholestasis was observed without marked inflammatory, fibrotic or steatotic histopathological changes (Fig. 1). Moreover, the typical histopathological picture of steatohepatitis or fibrocholestatic hepatitis was not seen in any of these subjects, neither was there evidence for biliary obstruction or cholestatis due to sepsis. In the control group of patients with stable liver function, only minimal cholestatis was found in a single patient.

Table 2 Serological and histopathological data of the study patients. (interestingly, as the authors say, histopathology markers of the advanced disease patients & the stable control group patients were similar for markers of MHAI inflammatory, piecemeal necrosis, bridging necrosis, lobular inflammation, portal inflammation, MHAI fibrosis, lobular fibrosis, iron deposition; but worse in advanced patients for cholestais (mean 1.28 vs 0.14). Tables below will be posted in article archived on NATAP website. In the Biochemistry table below, total bilirubin (9.5 vs 1.19 mg/dL) and alkaline phosphatase (413 vs 110 IU/mL) were worse for advanced patients than for control patients.

Liver specimens obtained by percutaneous liver biopsy (Menghini technique) at the University of Bonn were retrospectively analyzed in seven HIV-HCV co-infected male patients, all of whom had died from liver failure within a maximum of 6 months after the biopsy and in whom no other cause of liver disease than chronic HCV infection had been identified. Liver specimens had been taken for routine diagnostic purposes when patients suffered from unexplained deterioration of their liver disease with jaundice, fatigue, ascites and encephalopathy and failing biosynthetic hepatic functions. Seven HIV-HCV co-infected patients with stable immune function, in whom liver biopsies were taken prior to planned interferon therapy, were chosen as a reference group for histological comparison. Patients of this control group had been selected prior to histological evaluation as a match for patients with fatal liver disease with respect to the documented duration of HIV and HCV infection. Patients with a history of alcohol abuse or liver diseases other than chronic HCV infection were not included in the present study.

None of the patients had evidence of extrahepatic bile flow obstruction as determined by ultrasound and/or cholangiography. Patency of the hepatic arteries was proven by Doppler ultrasound examination. In all patients meticulous biochemical and microbiological studies had excluded evidence of autoimmune or metabolic liver disease as well as opportunistic liver diseases including infections by cytomegalovirus (CMV), Epstein-Barr virus or herpes viruses. All 14 patients were hepatitis B surface antigen-negative. In addition, none of our patients had received drugs associated with a known potential for cholestatic liver injury. The study was conducted in accordance with the institutional review board of the University of Bonn and conformed to the declaration of Helsinki from 1975.

The diagnosis of hepatitis C infection was based on the detection of HCV-antibodies by use of the microparticle enzyme immunoassay (MEIA) (Axsym, Abbott, Wiesbaden, Germany). The HCV-RNA was quantified with the nucleic acid purification kit (Viral Kit, Qiagen, Hilden, Germany) followed by a reverse transcription and a nested polymerase chain reaction (Amplicor HCV Monitor Test Version 2.0, Roche, Basel, Switzerland). The diagnosis of HIV infection was established by a positive HIV-1-specific ELISA test (Abbott, Wiesbaden, Germany) and confirmed by a positive HIV-1 immunoblot (Bio-Rad, Munich, Germany).

Liver specimens were routinely processed and stained with hematoxylin-eosin. Periodic acid-Schiff (PAS) and Grocott stains as well as immunohistochemical studies with antibodies directed against CMV-early antigen (monoclonal antibody) and ubiquitin (polyclonal antibody; both antibodies form Dako, Hamburg Germany) were also performed using the avidin-biotin-enzyme complex (ABC) method. Two experienced pathologists, who were blinded to all clinical information other than HCV and HIV seropositivity, assessed histological findings semiquantitatively via established scores. The degree of inflammation was graded and the amount of fibrosis was staged using the Ishak modified hepatic activity index (MHAI index). 12 The severity of liver steatosis and iron deposition was assessed in analogy to the classification system of Brunt et al. 13 Centrilobular fibrosis and cholestasis were evaluated by a separate score, ranging from none (0) to mild (1), moderate (2) or severe (3).

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