Assessment of genotype and molecular evolution of hepatitis C virus in formalin-fixed paraffin-embedded liver tissue from patients with chronic hepatitis C virus infection

Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5'non-coding region (5'NCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5'NCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5'NCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5'NCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5'NCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV.     

Serum hepatitis C virus RNA levels and histologic findings in liver allografts with early recurrent hepatitis C

BACKGROUND: Histopathologic features of early recurrent hepatitis C after orthotopic liver transplantation (OLTx) may be modified by immunosuppressive therapy or complicated by other conditions. Hepatitis C virus (HCV) RNA level usually increases after OLTx, but its correlation to histologic findings is not clear. OBJECTIVE: To evaluate the histologic findings of early recurrent hepatitis C in liver allografts and its correlation to serum HCV RNA level. METHODS: We studied 14 patients who underwent OLTx for chronic HCV infection. Thirty liver biopsy specimens and HCV RNA levels of 22 corresponding plasma samples obtained during the first 6 months following OLTx were analyzed. The control group (9 patients, 25 biopsy specimens) was chosen at random from patients with chronic liver disease other than HCV who were undergoing OLTx, and all tested negative for HCV RNA by polymerase chain reaction after OLTx. RESULTS: Statistically significant pathological features of early recurrent HCV infection were the number of acidophilic bodies, piecemeal necrosis, lymphocyte predominance in the portal tracts, and fibrous septum. These findings and histologic activity index scores increased with time after OLTx. The HCV RNA levels determined by branched DNA assay showed no significant correlation with histologic features. However, patients with higher histologic activity index scores tended to have higher RNA levels. CONCLUSIONS: Liver biopsy specimens are helpful for the diagnosis or confirmation of early recurrent hepatitis C in liver allografts, but serial biopsy specimens are sometimes required for definite diagnosis. The HCV RNA levels are usually higher in patients who display signs of more severe liver damage.     

Recurrence of clinically significant hepatitis A following liver transplantation for fulminant hepatitis A.

BACKGROUND: We report hepatitis A virus (HAV) infection of a liver allograft following transplantation for fulminant liver failure due to HAV infection. This rare condition has been described in only three patients to date. After liver transplantation allograft function was good, but starting 80 days after transplantation, episodes of acute graft dysfunction were observed. OBJECTIVES: To elucidate the reason for acute hepatic dysfunction a large number of differential diagnoses were tested. RESULTS: HAV RNA was undetectable for more than 80 days after transplantation. Detection of genomic HAV RNA by RT-PCR in serum and stool at the time of graft dysfunction led to the diagnosis of recurrent HAV infection. CONCLUSIONS: We suggest that the risk of HAV reinfection after liver transplantation may be far higher than expected as results may be misinterpreted as rejection episodes.     

Impact of alloimmune T cell responses on hepatitis C virus replication in liver transplant recipients

We investigated the influence of alloimmune T cell responses on hepatitis C virus (HCV) replication in HCV-infected patients after liver transplantation (LT). To monitor the immune-status in 27 HCV-infected LT recipients, we routinely performed mixed lymphocyte reaction (MLR) assays within 4 weeks after LT. HCV RNA titers in most patients fluctuated in inverse proportion to the stimulation index (SI) of anti-donor reactive T cells early after LT. Two weeks after LT, recipients with high HCV RNA titers (>1000 KIU/mL) displayed a significantly lower SI for anti-donor reactive T cells than recipients with low HCV RNA titers did (<1000 KIU/mL). An in vitro transwell assay mimicking the anatomical features of the interaction between HCV-infected hepatocytes and alloreactive T cells in allograft livers demonstrated that interferon (IFN)-γ was necessary to suppress HCV replication. This study proves the significant impact of alloimmune T cell responses on HCV replication in HCV-infected LT recipients.     

A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells

Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 μg liver RNA and 10⁷–10⁸ copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10⁹ copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.     

Immunohistochemical assessment of hepatitis C virus antigen in cholestatic hepatitis after liver transplantation

BACKGROUND: Patients with common variable immunodeficiency may exhibit rapidly progressive hepatitis when infected with hepatitis C virus (HCV), leading to cirrhosis and liver failure. Liver transplantation in these patients may result in a cholestatic form of HCV reinfection with exceptionally high virus loads. AIMS: To report an immunohistochemical investigation of the pretransplant and post-transplant liver of one such patient. METHODS/RESULTS: On immunohistochemical staining of frozen sections with anti-HCV core monoclonal antibody or fluorescein labelled human polyclonal anti-HCV IgG, no HCV antigens were demonstrated in the native cirrhotic liver removed at transplant, despite a viral load of 10(6.4) genomes/g. The transplanted liver, collected six weeks post-transplant, exhibited cholestatic recurrent hepatitis, had an HCV virus load of 10(10) genomes/g of liver, and revealed HCV antigen in the cytoplasm of most hepatocytes, with a pronounced periportal distribution. No virus antigen was demonstrable in other cell types. The core antigen was also detected in paraffin wax embedded, formaldehyde fixed tissue of this liver after high temperature antigen retrieval, but not in the native cirrhotic liver or a selection of HCV positive livers collected pretransplant from immunocompetent patients. Attempts to delineate the distribution of E1, NS3, and NS4 antigens were unsuccessful because monoclonal antibodies to these antigens produced "false positive" staining of foci of hepatocytes in the post-transplant livers of HCV seronegative patients with cholestasis. CONCLUSION: This case provided an opportunity to study the natural development of HCV during acute infection in the absence of an immune response, and may help to elucidate the pathogenesis of HCV recurrence in liver allografts.     

Hepatitis C-associated granulomas after liver transplantation: morphologic spectrum and clinical implications

Liver granulomas have been described in biopsy specimens from people with de novo chronic hepatitis C virus (HCV) infection and in allograft biopsy specimens from recipients of transplants for HCV-related liver disease. The latter have not been well studied, and there are no data regarding the prevalence, morphologic spectrum, and clinical significance of HCV-associated granulomas after liver transplantation. We observed granulomas in allograft liver biopsy specimens of 4 (8%) of 53 recipients of transplants for HCV-related end-stage liver disease during a 3-year period. Initial appearance of granulomas ranged from 4 to 41 weeks after transplantation. Lobular and portal nonnecrotizing, epithelioid granulomas and lobular microgranulomas were observed, with the latter predominating. Serum transaminase and alkaline phosphatase levels were significantly higher in patients with granulomas than in age- and sex-matched control subjects, but histologic disease activity, cellular rejection scores, HCV genotypes, viral titers, and retransplantation rate owing to recurrent disease were not significantly different. Our study suggests that a granulomatous response to HCV infection occurs in a subset of patients after transplantation; however, this histologic finding does not portend a worse clinical outcome.     

Donor-specific hyporesponsiveness in ELISPOT assay is associated with early recurrence of hepatitis C in liver transplant recipients

Recurrence of hepatitis C in liver transplant recipients is a common event that often leads to loss of the allograft. There are no means to prevent, or even predict, those patients who are more prone to early aggressive recurrence. Therefore there is an increased need for tailored immunosuppression protocols specific to this patient population. Fifteen liver transplant recipients (eight hepatitis C virus [HCV]+; 7 HCV-) were followed for 12-24 months after transplantation. The frequency of donor-specific interferon (IFN)-gamma- or interleukin-10-producing lymphocytes was monitored using ELISPOT assays. Of the eight HCV+ recipients, six experienced recurrence within the first year after transplant. All six patients had very low (negligible) frequency of donor-specific IFN-gamma precursors; in most cases not higher than the nonstimulated (spontaneous) secretion rate. The other two patients who did not recur exhibited donor-specific IFN-gamma reactivity. A significant difference in the frequency of alloantigen-specific T cells was observed between HCV+ recipients and patients with other indications for transplantation. The results of this preliminary study suggest that posttransplant monitoring of the frequency of donor-specific IFN-gamma-producing precursors may differentiate a subset of patients at risk for early recurrent hepatitis C and therefore may help to devise treatment strategies for HCV+ liver recipient after transplantation.     

Evolution of hepatitis B virus liver disease after hepatic replacement. Practical and theoretical considerations

The morphologic evolution of hepatitis B virus (HBV) liver disease in 45 hepatic allograft recipients who were HBV surface-antigen positive (HBs-Ag+) at the time of liver replacement and who survived for more than 60 days was studied by routine histologic and immunocytochemical analysis of serial pathology specimens. The findings in these patients were compared to a control group of 30 individuals who were immune to the HBV (anti-HBs antibody positive), but required hepatic replacement for other reasons. Eight of the forty-five (18%) HBsAg-positive patients have no serologic evidence of HBV reinfection after transplantation. All 37 remaining patients are reinfected; 21 (47%) developed chronic active hepatitis and/or cirrhosis, 3 (7%) developed submassive necrosis, and 6 (14%) developed chronic lobular hepatitis. One patient lost her graft to chronic rejection, despite reinfection with the B virus. Four other patients (9%) developed a chronic carrier state. No long-term follow-up biopsies were available in the remaining two patients. The histologic features associated with dysfunction related to recurrent HBV infection evolved from an acute to chronic phase and were similar to hepatitis B seen in nonallografted livers. Furthermore HBV-related lesions could be separated from rejection using routine histology alone. The only exception to this conclusion was the occurrence of a peculiar HBV-related lesion in two recipients, described herein. Immunohistochemical analysis demonstrated the presence of viral antigens in almost all cases. Hepatic inflammation also was commonly present during HBV disease and consisted mostly of accessory cells and T lymphocytes. Analysis of the effect of major histocompatibility complex matching revealed no clear association between the number of class I or II matches or mismatches and the development, or pattern, of active hepatitis in the allograft. Peculiar pathologic alterations in several of the biopsies and failed allografts after HBV reinfection suggests that, under special circumstances, the B virus may be cytopathic.     

HCV in liver transplantation

HCV-related cirrhosis represents the leading indication for liver transplantation in the Western countries. HCV reinfection after liver transplantation occurs in virtually all patients transplanted for HCV-related liver disease Histological evidence of chronic HCV infection develops in 50 to 90 % of patients by 12 months after liver transplantation, and cirrhosis occurs in about 20 % of patients within 5 years after transplant. Several studies have evaluated host, viral, and transplant-related factors that might be associated with the severity of HCV recurrence. Among host factors, immunosuppression is one of the major factors that accounts for accelerated HCV recurrence and it has been an area of extensive research and controversy. Donor age, steatosis, and immunogenetic factors are also relevant in determining the outcome in patients transplanted for HCV-related cirrhosis. A major step to prevent complications of HCV recurrence related to the rapid fibrosis is the posttransplant antiviral treatment. Two strategies have been tried: pre-emptive or other strategies as soon as possible after liver transplantation or elective therapy once there is histological evidence of recurrent hepatitis C. Retransplantation due to graft failure from recurrent hepatitis C is rarely an option in the era of organ shortage as it is associated with poor outcome, but many case needs to be considered early in the evolution of disease. New antivirals may change the outcome dramatically of patients transplanted for HCV cirrhosis.     

Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction.

Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.     

Agreement in pathologic interpretation of liver biopsy specimens in posttransplant hepatitis C infection

Hepatitis C virus-related disease is rapidly becoming the most common indication for orthotopic liver transplant (OLT) in the United States. Although post-OLT hepatitis C viremia is universal, 40% to 60% of patients develop recurrent chronic hepatitis C. Distinguishing recurrent chronic hepatitis C infection from acute rejection may be difficult because of overlapping histopathologic features. To improve our diagnostic accuracy we undertook a study to determine interobserver and intraobserver agreement between pathologists examining post-OLT liver biopsy specimens in patients from our transplant database. Clinical data and microscopic sections from 26 patients with hepatitis C virus-related OLT were reviewed. Biopsy specimens were obtained because of abnormal liver enzymes (21/26) or routine post-OLT follow-up (5/26), representing both early (18+/-11 days) and late (252+/-206 days) post-OLT periods. Unidentified sections were examined by an experienced pathologist in a randomly assigned order and reexamined 6 weeks later in the same fashion by the initial reviewer and a second experienced pathologist. Interobserver and intraobserver agreement was calculated using K statistic. The intraobserver agreement was 81 % with a kappa coefficient of 0.67 (P = .001). The interobserver agreement was 78% with a kappa coefficient of 0.60 (P < .001). The early post-OLT biopsy specimens (18+/-11 days) were the most difficult to interpret.     

Recurrent hepatitis B in liver allograft recipients. Differentiation between viral hepatitis B and rejection.

The histologic findings in the original liver obtained from 9 liver allograft patients with active B virus hepatitis were compared with 28 posttransplant pathology specimens. All specimens were studied with the use of light and immunohistochemical microscopy in conjunction with pertinent clinical data. Eight of the 9 patients had chronic active hepatitis B (HB) with cirrhosis, prior to transplant, one of which had coexistent hepatocellular carcinoma. The ninth patient had fulminant hepatic necrosis secondary to acute HB prior to transplantation. In all of the patients with chronic HB prior to transplantation who survived more than 2 months after transplantation recurrent infection of the graft developed despite perioperative HB immunoglobulin therapy. The patient with acute fulminant hepatitis B pretransplant has done well postoperatively and has evidence of HB virus immunity (positive anti-HBs) 15 months after transplantation. Examination of tissue specimens obtained during episodes of allograft dysfunction in these 9 patients indicate that pathologic alterations of active HB infection of the allograft are associated with a preferential lobular insult, whereas those occurring in rejection preferentially involve portal tract structures. Serologic data combined with biopsy histopathologic data are essential in distinguishing between the two quite different events.     

Dynamics of Defective Hepatitis C Virus Clones in Reinfected Liver Grafts in Liver Transplant Recipients: Ultradeep Sequencing Analysis

Hepatitis C virus (HCV) reinfects liver allografts in transplant recipients by replicating immediately after transplantation, causing a rapid increase in blood serum HCV RNA levels. We evaluated dynamic changes in the viral genetic complexity after HCV reinfection of the graft liver; we also identified the characteristics of replicating HCV clones using a massively parallel ultradeep sequencing technique to determine the full-genome HCV sequences in the liver and serum specimens of five transplant recipients with genotype 1b HCV infection before and after liver transplantation. The recipients showed extremely high genetic heterogeneity before transplantation, and the HCV population makeup was not significantly different between the liver and blood serum specimens of the individuals. Viral quasispecies complexity in serum was significantly lower after liver transplantation than before it, suggesting that certain HCV clones selectively proliferated after transplantation. Defective HCV clones lacking the structural region of the HCV genome did not increase in number, and full-genome HCV clones selectively increased in number immediately after liver transplantation. A re-increase in the same defective clone existing before transplantation was detected 22 months after transplantation in one patient. Ultradeep sequencing technology revealed that the genetic heterogeneity of HCV was reduced after liver transplantation. Dynamic changes in defective HCV clones after liver transplantation indicate that these clones have important roles in the HCV life cycle.     

Expression of the interferon-inducible proteins MxA and IFI16 in liver allografts

Aims:  To test the hypothesis that the activation of the interferon (IFN) system pathways might link hepatitis C virus (HCV) recurrence in the liver allograft with acute cellular rejection.
Methods and results:  In this retrospective study, allograft biopsy specimens from 28 adult patients (14 HCV+ and 14 HCV−) who had undergone their first liver transplantation were analysed. Eleven biopsy specimens showed acute cellular rejection (Banff rejection activity index score ≥3). Specimens were immunostained for two IFN-inducible proteins, MxA and IFI16, and for CD45. The predominant MxA reactivity pattern was hepatocytic, whereas IFI16 was expressed in both the hepatocellular and inflammatory compartments. Moderate to strong MxA expression in hepatocytes was associated positively with rejection score ( P  < 0.01), donor's age ≤45 years ( P  < 0.05) and aspartate aminotransferase levels >40 U/l on the day of biopsy ( P  < 0.05), and inversely with infiltration of portal triads by IFI16+/CD45+ cells ( P  < 0.005) and time to progression beyond Ishak stage 2 of recurrent hepatitis C ( P  < 0.01). On multivariate analysis, MxA expression in hepatocytes was independently associated with allograft rejection and donor's age.
Conclusions:  Acute allograft rejection and recurrence of HCV infection in the liver allograft appear to intersect in the IFN system pathways.