Marek Radkowski

Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C.

It is unclear whether the current antiviral treatment for chronic hepatitis C virus (HCV) infection results in complete elimination of the virus, or whether small quantities of virus persist. Our study group comprised 17 patients with chronic HCV who had sustained virological response (SVR) after interferon/ribavirin treatment. Serum and peripheral blood mononuclear cells were collected 2 to 3 times at 3‐ to 6‐month intervals starting 40 to 109 months (mean, 64.2 ± 18.5 months) after the end of therapy. In addition, lymphocyte and macrophage cultures were established at each point. In 11 patients, frozen liver tissue samples were available from follow‐up biopsies performed 41 to 98 months (mean, 63.6 ± 16.7 months) after therapy. Presence of HCV RNA was determined by sensitive reverse‐transcriptase polymerase chain reaction, and concentration of positive and negative strands was determined by a novel quantitative real‐time reverse transcriptase polymerase chain reaction. Only 2 of 17 patients remained consistently HCV RNA negative in all analyzed compartments. HCV RNA was detected in macrophages from 11 patients (65%) and in lymphocytes from 7 patients (41%). Viral sequences were also detected in 3 of 11 livers and in sera from 4 patients. Viral replicative forms were found in lymphocytes from 2 and in macrophages from 4 patients. In conclusion, our results suggest that in patients with SVR after therapy, small quantities of HCV RNA may persist in liver or macrophages and lymphocytes for up to 9 years. This continuous viral presence could result in persistence of humoral and cellular immunity for many years after therapy and could present a potential risk for infection reactivation. (HEPATOLOGY 2005;41:106–114.)

Search for Hepatitis C Virus Negative-Strand RNA Sequences and Analysis of Viral Sequences in the Central Nervous System: Evidence of Replication

ABSTRACT Patients with chronic hepatitis C are more likely to have significant changes in their physical and mental well-being than patients with liver disease of other etiology, and hepatitis C virus (HCV) has been occasionally implicated in diseases of the central nervous system. We analyzed the presence of the HCV negative-strand RNA sequence, which is the viral replicative intermediary, in autopsy brain tissue samples from six HCV-infected patients. Negative-strand HCV RNA was searched for by a strand-specific Tth-based reverse transcriptase PCR, and viral sequences amplified from brain tissue and serum were compared by single-strand conformational polymorphism analysis and direct sequencing. HCV RNA negative strands were detected in brain tissue in three patients. In two of these patients, serum- and brain-derived viral sequences were different and classified as belonging to different genotypes. In one of the latter patients, HCV RNA negative strands were detected in lymph node and, while being different from serum-derived sequences, were identical to those present in the brain. The results of the present study suggest that HCV can replicate in the central nervous system, probably in cells of the macrophage/monocyte lineage.

Outcome of liver transplantation in hepatitis C virus–infected patients who received hepatitis C virus–infected grafts

BACKGROUND & AIMS The present organ shortage has brought into question the suitability of hepatitis C virus (HCV)-positive grafts. This study reviewed the outcome of such transplantations in our institution. METHODS Twenty-three HCV-positive patients who underwent orthotopic liver transplantation (OLT) for end-stage liver disease with HCV-positive grafts in 1992-1995 were studied. Only patients who survived more than 30 days were included in the analysis. Control group included 169 patients who underwent transplantation for HCV-related cirrhosis and received HCV-negative organs. RESULTS Patients who received HCV-infected organs had a cumulative survival rate of 89% and 72% at 1 and 5 years, respectively, vs. 88% and 73% for the control group (NS). There was no difference in graft survival, incidence of cirrhosis, mean hepatitis activity index score, fibrosis, or mean activity of serum transaminases. There was a trend toward lower incidence of recurrent hepatitis C in the study group compared with control (21% vs. 23% at 1 year and 47% vs. 64% at 5 years; NS). Patients in whom the donor strain became predominant after transplantation had significantly longer disease-free survival than patients who retained their own HCV strain (P < 0.003). CONCLUSIONS HCV-infected livers transplanted into HCV-infected recipients do not appear to convey a worse outcome in the initial years after OLT than HCV-negative grafts.

Hepatitis C Virus Neuroinvasion: Identification of Infected Cells

ABSTRACT Hepatitis C virus (HCV) infection often is associated with cognitive dysfunction and depression. HCV sequences and replicative forms were detected in autopsy brain tissue and cerebrospinal fluid from infected patients, suggesting direct neuroinvasion. However, the phenotype of cells harboring HCV in brain remains unclear. We studied autopsy brain tissue from 12 HCV-infected patients, 6 of whom were coinfected with human immunodeficiency virus. Cryostat sections of frontal cortex and subcortical white matter were stained with monoclonal antibodies specific for microglia/macrophages (CD68), oligodendrocytes (2′,3′-cyclic nucleotide 3′-phosphodiesterase), astrocytes (glial fibrillary acidic protein [GFAP]), and neurons (neuronal-specific nuclear protein); separated by laser capture microscopy (LCM); and tested for the presence of positive- and negative-strand HCV RNA. Sections also were stained with antibodies to viral nonstructural protein 3 (NS3), separated by LCM, and phenotyped by real-time PCR. Finally, sections were double stained with antibodies specific for the cell phenotype and HCV NS3. HCV RNA was detected in CD68-positive cells in eight patients, and negative-strand HCV RNA, which is a viral replicative form, was found in three of these patients. HCV RNA also was found in astrocytes from three patients, but negative-strand RNA was not detected in these cells. In double immunostaining, 83 to 95% of cells positive for HCV NS3 also were CD68 positive, while 4 to 29% were GFAP positive. NS3-positive cells were negative for neuron and oligodendrocyte phenotypic markers. In conclusion, HCV infects brain microglia/macrophages and, to a lesser extent, astrocytes. Our findings could explain the biological basis of neurocognitive abnormalities in HCV infection.

Detection and Analysis of Hepatitis C Virus Sequences in Cerebrospinal Fluid

ABSTRACT Hepatitis C virus (HCV) sequences were detected in cerebrospinal fluid (CSF) in 8 of 13 HCV-positive patients. In four patients harboring different virus strains in serum and peripheral blood mononuclear cells (PBMC), CSF-derived virus was similar to that found in PBMC, which suggests that PBMC could carry HCV into the brain.

Uneven Distribution of Hepatitis C Virus Quasispecies in Tissues from Subjects with End-Stage Liver Disease: Confounding Effect of Viral Adsorption and Mounting Evidence for the Presence of Low-Level Extrahepatic Replication

ABSTRACT We have found differences among the populations of hepatitis C virus sequences in serum, peripheral blood mononuclear cells (PBMCs), and various tissues in patients with chronic hepatitis C. These results are compatible with the existence of independent viral compartments in the infected host. Our results also suggest that PBMCs, and probably various tissues, can selectively adsorb viral subpopulations differing in the E2 region.

Hepatitis C virus negative strand RNA is not detected in peripheral blood mononuclear cells and viral sequences are identical to those in serum: a case against extrahepatic replication.

Peripheral blood mononuclear cells (PBMCs) from 27 hepatitis C virus (HCV)-infected patients were analysed for the presence of HCV negative strand RNA with strand-specific Tth-based RT-PCR. No negative strand RNA was detected in any sample, and positive strand HCV sequences amplified from PBMCs were identical to those found in serum. These findings suggest that HCV does not replicate in PBMCs, and the presence of HCV sequences at this site is compatible with passive virus adsorption and/or contamination by circulating virus.

The Presence of Active Hepatitis C Virus Replication in Lymphoid Tissue in Patients Coinfected with Human Immunodeficiency Virus Type 1

The existence of extrahepatic replication sites of hepatitis C virus (HCV) remains controversial. Highly strand-specific Tth-based reverse transcriptase-polymerase chain reaction was used to search for the presence of viral RNA negative strand in lymph nodes from 16 patients with AIDS and in peripheral blood mononuclear cells (PBMC) from 14 other human immunodeficiency virus (HIV)-positive patients. Negative-strand HCV RNA was detected in lymph node samples from 10 patients (63%) and in PBMC from 5 (36%). This suggests that, at least under circumstances of impaired immunity associated with HIV infection, HCV is lymphotropic in vivo. However, the clinical implications of these findings need to be further investigated.

Activation of brain macrophages/microglia cells in hepatitis C infection

Objectives Hepatitis C virus (HCV) infection is commonly associated with cognitive dysfunction. Viral sequences and proteins were previously found in brain macrophage/microglia cells. The aim of the current study was to determine whether HCV infection affects the expression of key cytokines and chemokines in these cells. Methods Autopsy brain tissue from 15 patients was studied; 7 patients were HCV positive and 8 were HCV negative. Cryostat sections of frontal cortex and subcortical white matter were stained with monoclonal antibodies specific for microglia/macrophages (anti-CD68) and separated by laser capture microscopy. Transcripts representing 25 various cytokines and chemokines were measured by real-time quantitative PCR. Results Compared with HCV-negative controls, HCV-positive patients demonstrated significantly higher levels of proinflammatory cytokines interleukin 1α (IL-1α), IL-1β, tumour necrosis factor α (TNFα), IL-12 and IL-18. HCV infection was also associated with increased transcription of chemokines IL-8, IL-16 and interferon-inducible protein 10 (IP-10). Type 1 interferon (IFN) activation was suggested by increased concentrations of IFNβ and myxovirus resistance protein A (MxA) transcripts. Similar results were obtained when CD68-positive/HCV-positive cells were compared with CD68-positive/HCV-negative cells in each of the 7 HCV-infected patients. Conclusion Evidence was found for activation of brain macrophages/microglia cells in autopsy brain tissue from HCV-positive patients. These findings could relate to the common presence of neurocognitive dysfunction among patients with chronic hepatitis C.

Negative-Strand Hepatitis C Virus (HCV) RNA in Peripheral Blood Mononuclear Cells from Anti-HCV–Positive/HIV-Infected Women

BACKGROUND Hepatitis C virus (HCV) has been reported to replicate in peripheral blood mononuclear cells (PBMCs), particularly in patients coinfected with HCV and human immunodeficiency virus (HIV). However, there are limited data regarding the prevalence of and the factors associated with extrahepatic replication. METHODS The presence of negative-strand HCV RNA in PBMCs was evaluated by a strand-specific assay for 144 anti-HCV-positive/HIV-infected women enrolled in the Womens Interagency HIV Study. One to 5 PBMC samples obtained from each woman were tested. Multivariate analyses were used to assess for associations with the clinical and demographic characteristics of the women. RESULTS Negative-strand HCV RNA was detected in 78 (25%) of 315 specimens, and, for 61 women (42%), > or = 1 specimen was found to have positive results. The presence of negative-strand HCV RNA in PBMCs was significantly positively associated with an HCV RNA plasma level of > or = 6.75 log copies/mL (P=.04) and consumption of > or = 7 alcoholic drinks per week (P=.02). It was also negatively associated with injection drug use occurring in the past 6 months (P=.03). A negative association with a CD4+ CD38+ DR+ cell percentage of > 10% and a positive association with acquired immunodeficiency syndrome were borderline significant (P=.05). CONCLUSIONS HCV replication in PBMCs is common among HIV-coinfected women and appears to be a dynamic process related to lifestyle, virologic, and immunologic factors.