Debra Adair

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.

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.

Evidence for viral persistence in patients who test positive for anti-hepatitis C virus antibodies and have normal alanine aminotransferase levels.

It is unclear whether patients who test positive for anti-hepatitis C virus (HCV) antibodies and have normal alanine aminotransferase (ALT) levels remain infected with the virus. Eleven patients who tested positive for anti-HCV antibodies, had persistently normal ALT levels, and tested negative for HCV RNA by commercial test were studied. Serum and peripheral blood mononuclear cells (PBMCs) were collected 2-3 times at 3-6-month intervals, and PBMCs were cultured with phytohemagglutinin and pokeweed mitogen. HCV RNA was detected in serum samples from 6 (55%) and in PBMCs from 11 (100%) patients. Our results suggest that, in asymptomatic patients who test positive for anti-HCV antibodies, small quantities of HCV RNA commonly persist, even in patients who test negative for HCV RNA in serum by commercial tests.

Hepatitis C Virus Quasi-Species Dynamics Predict Progression of Fibrosis after Liver Transplantation

BACKGROUND The dynamics of hepatitis C virus (HCV) quasi species in the E2 region may correlate with the course of infection after orthotopic liver transplantation (OLT). METHODS Thirty-four patients who underwent transplantation for HCV-related cirrhosis were studied. Serum and liver samples were available before OLT and at 1 week, 4 months, and 1 year after OLT. Patients were divided into group 1 (Knodell/Ishak fibrosis stage [FS] at 1 year, <2) and group 2 (FS at 1 year, > or =2). Complexity was estimated by the number of bands in a single-strand conformational polymorphism assay, whereas diversity was measured by Shannon entropy (SE) and median mobility shift (MMS) values derived from the heteroduplex mobility assay. Diversity dynamics were measured at transmission (before OLT vs. 1 week after OLT) and after OLT (1 week after OLT vs. 1 year after OLT). RESULTS Complexity was higher in group 1 patients than in group 2 patients before OLT (P<.02) and at 1 week after OLT (P<.04). Diversity decreased in group 1 at transmission, as measured by either SE (P<.01) or MMS (P<.04). However, diversity increased in this group after OLT, as measured by SE (P<.03) or MMS (P<.02). FS at 1 year after OLT correlated with transmission changes, as measured by SE (r=0.642, P<.0001) and MMS (r=0.443, P<.04), and with post-OLT changes (for SE: r=-0.583, P<.01; for MMS: r=-0.536, P<.01). CONCLUSIONS HCV complexity and diversity in the E2 region correlated with the severity of recurrence of HCV infection after OLT. Increased diversity of quasi species at transmission correlated with a higher FS at 1 year. However, increased diversity of quasi species in the post-OLT period correlated with a lower FS at 1 year. The dynamics of HCV quasi species in patients who undergo transplantation are predictive of outcome.

Differential display analysis of gene expression in brains from hepatitis C-infected patients.

Objectives:Hepatitis C virus (HCV) infection is often associated with cognitive dysfunction, fatigue and depression. The current study was undertaken to determine whether HCV infection affects gene expression in brain tissue. Design:We analysed the gene expression pattern in brain tissue in a group of HCV-infected patients compared with HCV-negative controls. Methods:Brain tissue samples were obtained at autopsy from three HCV-positive patients and three HCV-negative control patients. The analysis of gene expression was conducted using differential display and reverse Northern hybridization. Only those genes that were up or downregulated more than 1.8 times were considered to be differentially expressed. Results:Altogether, 29 differentially expressed genes were identified by differential display and subsequently confirmed by reverse Northern hybridization. A prominent finding was the downregulation of mitochondrial oxidative phosphorylation genes in HCV-infected patients. The impairment of brain oxidative/energy metabolism has previously been suggested to be the proximate cause of many disorders that impair mentation. Another finding was the downregulation of some ribosomal protein genes and several genes involved in transcription regulation, perhaps reflecting reduced metabolic activities. Conclusion:Our findings suggest for the first time that there may be a biological basis for the neuropsychiatric symptoms and cognitive impairment associated with HCV infection.

546 Infection of primary human macrophages with hepatitis C virus in vitro: Induction of tumor necrosis factor alpha and interleukin 8

Hepatitis C virus (HCV) has been reported to replicate in monocytes/macrophages in infected patients. However, it is unclear whether macrophages are susceptible to infection in vitro and whether such an infection is consequential. Sera from 26 HCV-infected patients were incubated with primary human macrophages collected from healthy donors. Virus negative strand was detected by a Tth enzyme-based strand-specific assay and virus sequences were analysed by single strand conformation polymorphism (SSCP) and sequencing. Concentrations of the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12p70 were measured in culture supernatants and respective mRNAs were analysed in cell extracts by quantitative RT-PCR. For 15 sera, HCV RNA was detectable in 2- and 3-week cultures from at least one donor. Virus negative strand was detected in 29 % of macrophage samples in this group. In four cases, HCV RNA sequences amplified from macrophages differed from those amplified from sera suggesting evolution during infection. Concentrations of TNF-alpha and IL-8 were found to be significantly higher in supernatants from HCV-infected cultures. In conclusion, these preliminary data suggest that primary human macrophages are susceptible to HCV infection in vitro and this infection is associated with the induction of cytokines TNF-alpha and IL-8.