Tomasz Laskus

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.

Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B

BACKGROUND & AIMS It was recently reported that two point mutations within the hepatitis B virus (HBV) core promoter region (A to T at position 1762 and G to A at position 1764) are associated with fulminant hepatitis and lead to hepatitis B e antigen (HBeAg)-negative phenotype. The aim of this study was to correlate core promoter sequence variations with HBeAg status and clinical outcome in various forms of HBV infection. METHODS Core promoter region of HBV was amplified by polymerase chain reaction and directly sequenced in 94 patients: 37 patients with fulminant hepatitis, 20 with acute self-limited hepatitis, 30 with chronic hepatitis, and 7 patients with end-stage cirrhosis. RESULTS Core promoter region was found to be heterogenous and no specific changes correlated with HBeAg/anti-HBeAg status or survival in patients with fulminant hepatitis. Substitutions at positions 1762 and 1764 were found in HBV strains from 4 patients (10%) with fulminant hepatitis, 2 patients (10%) with self-limited hepatitis, 8 patients (27%) with chronic hepatitis, and in 5 of 7 patients with end-stage cirrhosis. The majority of these patients were HBeAg positive. CONCLUSIONS Mutations at positions 1762 and 1764 are rarely observed in HBV strains from patients with fulminant hepatitis B in the United States but are common in patients with chronic hepatitis. Even when present, they seem to be insufficient to lead to the HBeAg-negative phenotype.

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.

Nucleotide sequence analysis of the precore region in patients with fulminant hepatitis B in the United States

BACKGROUND A precore defective hepatitis B virus (HBV) mutant unable to produce hepatitis B e antigen (HBeAg) has been associated with fulminant hepatitis B. We have studied the etiologic contribution of precore mutants among North American patients with this disorder. METHODS We studied 39 patients with fulminant hepatitis B. The precore and proximal core regions of HBV from 37 of 39 patients were sequenced. RESULTS Four patients (10.8%) harbored nonsense mutants likely to produce an HBeAg negative HBV infection; two such mutants had a G to A substitution at position 1896, one lost the precore initiation codon, and one harbored a stop codon immediately downstream of the precore initiation codon. Recovered sequences from seven additional patients displayed silent or missense mutations in these regions. All delta coinfected patients harbored known wild type strains of HBV. A significantly poorer survival was associated with antibody to HBe positivity and presence of nucleotide substitutions in the precore/core region. CONCLUSIONS The prevalence of precore mutations in 37 patients from the United States was lower than reported elsewhere; only two patients were found to have the G to A transition mutation in the precore region at position 1896. We conclude that HBeAg negative HBV mutants do not play a predominant etiologic role among North American patients with fulminant hepatitis B.

HEPATITIS C VIRUS GENOTYPES IN LIVER TRANSPLANT RECIPIENTS: IMPACT ON POSTTRANSPLANT RECURRENCE, INFECTIONS, RESPONSE TO INTERFERON-α THERAPY AND OUTCOME

BACKGROUND End-stage liver disease due to hepatitis C virus (HCV) is the most common indication for liver transplantation in U.S. veterans. We investigated the influence of HCV genotypes on the incidence and timing of recurrent HCV hepatitis, survival, infectious morbidity, and response to interferon-alpha therapy in this unique patient population. METHODS HCV genotype was determined by direct sequencing of the NS5 region of HCV with type-specific primers. RESULTS Genotype 1a (66%, 32/47) was the predominant genotype. Type 1b was found in 25% (12/47) of patients and type 2b was found in 9% (4/47). Histopathologically recurrent HCV hepatitis developed in 53% (25/47) of the patients after transplantation. This group included 45% (14/31) of the patients with type 1a, 67% (8/12) of the patients with type 1b, and 25% (1/4) of the patients with type 2b (P>0.5). The time to recurrence and the severity of HCV recurrence as defined by aminotransferase levels or Knodell scores were not different among the three genotypes. There was a trend toward a higher incidence of major infections in patients with type 1b (75%) versus type 1a (48%) and type 2b (50%) (P=0.11). The response to interferon-a therapy did not differ significantly among the genotypes. Mortality at 5 years was 16% (5/31) in patients with genotype 1a, 42% (5/12) in patients with genotype 1b, and 50% (2/4) in patients with genotype 2b (P=0.06). CONCLUSIONS The incidence, time to recurrence, and response to interferon-alpha therapy did not differ between the various genotypes in our liver transplant recipients. However, there was a trend toward higher infectious morbidity and overall mortality in patients with genotype 1b after transplantation.

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.