Peripheral Blood Mononuclear Cells Hepatitis C Virus Research Articles

Detection of Occult Hepatitis C Virus Infection in Egyptian Patients Who Achieved a Sustained Virologic Response to Direct-Acting Antiviral Agents.

Occult hepatitis C virus (HCV) infection (OCI) is diagnosed based on the detection of HCV-RNA in non-serum reservoirs, such as peripheral blood mononuclear cells (PBMCs) and/or hepatocytes with undetectable HCV-RNA in the serum. The current study was designed to shed more light on the presence of occult HCV in a population of cases who achieved an SVR after receiving treatments for HCV-infection and its significance. This cross-sectional study evaluated 111 chronic HCV patients treated at Theodor Bilharz Research Institute, Egypt and achieved a sustained virological response (SVR) 12 -24 weeks after treatment with Direct acting antiviral drugs (DAAs). The treatment lasted 12 or 24 weeks using generic medications including Sofosbuvir (SOF) 400 mg/day and Daclatasvir (DCV) 60 mg/day ± weight-based Ribavirin (RBV) 600-1000 mg/day. After achieving the SVR 12 -24 weeks, all patients were subjected to clinical examination and full laboratory investigations. All the candidates were assessed for fibrosis pre/post-treatment by transient elastography (Fibroscan©). Eighty-seven patients (78.4%) received dual therapy (SOF/DCV) and 24 patients (21.6%) received triple therapy (SOF/DCV/RBV). One hundred and seven patients received the regimen for 12 weeks (96.4%) and only four patients received the regimen for 24 weeks (3.6%). All patients were examined in terms of HCV RNA in plasma and PBMCs. Nine patients (8.1%) were positive for PBMCs HCV RNA. The presence of Occult HCV infection (OCI) was significantly correlated with age, level of AFP, and the degree of liver stiffness. The OCI was present in 8.1% of the patients who achieved an SVR 12 - 24 weeks. These patients were mostly aged and with elevated AFP and advanced fibrosis. Monitoring and follow-up of those patients may help to assess the outcomes.

Detection of hepatitis C virus RNA in the peripheral blood mononuclear cells in patients with chronic hepatitis C and its relationship with effect of anti-viral treatment

Objective To detect the change of hepatitis C virus (HCV) RNA in the peripheral blood mononuclear cells (PBMC) and serum of patients with chronic hepatitis C (CHC) during treatment with peg-interferon α-2a (Peg IFNα-2a) plus ribavirin (RBV), and to analyze the clinical significance of HCV RNA detection in PBMC. Methods The peripheral blood samples of 20 CHC patients who visited Department of Infectious Diseases in Guangzhou No.8 People's Hospital from June 2013 to December 2014, were collected during treatment with Peg IFNα-2a+ RBV at different time points (week 0, 2, 4, 12, 24, 36 and 48). Serum and PBMC were separated. Accurate fluorescence quantification assay (Cobas TaqMan real time polymerase chain reaction[PCR]) was used to detect HCV RNA level in serum, while real-time PCR and nest-PCR were applied to detect HCV RNA in PBMC. Categorical data were analyzed by χ2 test. Results Accurate fluorescence quantification of serum HCV RNA showed that HCV RNA level decline rapidly after treatment (F=148.06, P<0.01), and 18 patients achieved HCV RNA undetectable at week 12 of treatment. The positive rate of nest-PCR was higher than real-time PCR (all P<0.01). Comparison of HCV RNA levels in serum and PBMC from 20 cases found that, the clearance rate of HCV RNA in PBMC was postponed. Two patients whose HCV RNA in PBMC kept detectable relapsed at week 24 after end of treatment. Conclusions HCV RNA can be detected in PBMC of CHC patients and the positive rate of nest-PCR is higher than real-time PCR. Antiviral therapy is effective on HCV both inside and outside PBMC, but the clearance rate of HCV RNA in PBMC is postponed compared with that in serum. Slow clearance of HCV in PBMC may be a risk factor for relapse after end of treatment. Key words: Hepatitis C; Peripheral blood mononuclear cells; HCV RNA; Interferon

Dynamic changes in viral population structure and compartmentalization during chronic hepatitis C virus infection in children

Classic phylogenetic and modern population-based clustering methods were used to analyze hepatitis C virus (HCV) evolution in plasma and to assess viral compartmentalization within peripheral blood mononuclear cells (PBMCs) in 6 children during 3.2–9.6yr of follow-up. Population structure analysis of cloned amplicons encompassing hypervariable region 1 led to the distinction of two evolutionary patterns, one highly divergent and another one genetically homogeneous. Viral adaptability was reflected by co-evolution of viral communities switching rapidly from one to another in the context of divergence and stability associated with highly homogeneous communities which were replaced by new ones after long periods. Additionally, viral compartmentalization of HCV in PBMCs was statistically demonstrated, suggesting their role as a pool of genetic variability.Our results support the idea of a community-based structure of HCV viral populations during chronic infection and highlight a role of the PBMC compartment in the persistence of such structure.

Replication of hepatitis C virus in peripheral blood mononuclear cells in patients with chronic hepatitis C treated with pegylated interferon alpha and ribavirin

Hepatitis C virus (HCV) is a primarily hepatotropic virus, but hepatocytes are not the only localization of its replication. It is still unclear if extrahepatic HCV replication, measured as the detection of HCV RNA negative strand in peripheral blood mononuclear cells (PBMCs) before initiation of treatment, has an influence on therapy response. Detection of HCV RNA in extrahepatic sites for assessment of therapy efficacy is not routinely used in clinical practice. The aim of the study was to evaluate whether the replication of HCV in PBMCs affects the rate of sustained virological response (SVR). The study group comprised 55 patients with chronic hepatitis C, originally treatment naive. They were treated with pegylated interferon (PEG-IFN) alpha 2a and ribavirin, with the standard dosing schedule. Parallel serum samples for HCV RNA and PBMC samples for HCV RNA negative strand were obtained at baseline, at the end of treatment and 24 weeks after finishing therapy. Undetectable HCV RNA in serum at the end of therapy was found in 48 patients (87.3%), while 33 patients (60.0%) achieved sustained virological response (SVR) (51% for HCV genotype 1 and 78% for genotype 3, respectively). Fifteen individuals (31.3%) were relapsers. Factors associated with significantly higher rate of SVR were young age, mild or no fibrosis and infection with HCV genotype 3. HCV RNA negative strand in PBMCs before treatment was found in 21.8% (12 out of 55 patients). HCV RNA negative strand was detected at baseline more frequently in patients who later achieved SVR. Relapse appeared significantly more often in patients with negative strand at the end of therapy: in 2 out of 15 individuals compared to 0 out of 33 patients (p=0.03). Presence of negative HCV RNA strand in PBMCs before treatment may be suggested as a potential marker of good treatment response. Detection of negative strand at the end of therapy is a predictor of relapse.

Human Interferon Regulatory Factor 2 Gene Expression is Induced in Chronic Hepatitis C Virus Infection—A Possible Mode of Viral Persistence

The interferon regulatory factors (IRFs) are a family of transcription factors known to be involved in the modulation of cellular responses to interferons (IFNs) and viral infection. While IRF-1 acts as a positive regulator, IRF-2 is known to repress IFN-mediated gene expression. The increase in the IRF-1/IRF-2 ratio is considered as an important event in the transcriptional activation of IFN-α gene toward development of the cellular antiviral response. This study was performed to assess the expression of IRF mRNAs along with the expression level of IFN-α, its receptor (IFNAR-1), and the signal transduction factor (STAT-1) in treatment naive hepatitis C virus (HCV)-infected subjects. Thirty-five chronically infected (CHC) patients and 39 voluntary blood donors as controls were included in the study. Quantification of HCV-RNA (ribonucleic acid) and genotyping were done by real-time polymerase chain reaction (PCR) and hybridization assays, respectively, using patient's serum/plasma. In both controls and patients, the serum level of IFN-α and IFN-α was measured by flow cytometry. Target gene expressions were studied by retro-transcription of respective mRNAs extracted from peripheral blood mononuclear cells (PBMCs) followed by PCR amplification and densitometry. Minus-strand HCV-RNA as a marker of viral replication in PBMCs was detected by an inhouse PCR assay. Both IRF-1 and IRF-2 genes were significantly enhanced in CHC than in control subjects (P < 0.001). A significant positive correlation (r (2) = 0.386, P <0.01) was obtained between higher IRF-2 gene expression and increasing level of HCV-RNA. Chronically infected subjects (13%) harboring replicating HCV in PBMCs showed no significant differences in gene expressions than the subjects without HCV in PBMCs. Our findings indicate that HCV modulates host immunity by inducing IRF-2 gene to counteract IRF-1-mediated IFN-α gene expression. Since the IRF-2 gene is known to encode oncogenic protein, the role of IRF-2 in CHC patients developing hepatocellular carcinoma warrants further studies.

Quantitation of hepatitis C virus RNA in peripheral blood mononuclear cells in HCV‐monoinfection and HIV/HCV‐coinfection

Peripheral blood mononuclear cells (PBMCs) represent an extrahepatic hepatitis C virus (HCV) reservoir, the significance of which is unclear due to limited studies and varying test methodologies. In this study, a commercial viral load assay for measuring cell-associated PBMC HCV RNA was evaluated. HCV RNA was extracted from PBMCs, sorted CD14+, and CD19+ cells and corresponding plasma samples using the Abbott m2000 and Real-Time HCV assay. Test performance and influence of HIV seropositivity on plasma and PBMC HCV RNA were studied. Among 51 patients, 67 and 62 unique patient samples had detectable plasma and PBMC HCV viral load, respectively. The median PBMC viral load was 535 IU/1 M cells (range 29-5,190). CD19+ cells had significantly higher viral load than CD14+ cells (median log(10) HCV viral load 2.63 vs. 1.50 IU/ml; P< 0.001). Stability of PBMC viral load over time was demonstrated in untreated patients; all patients with an undetectable plasma HCV viral load after HCV treatment also demonstrated undetectable PBMC viral load. Repeated testing in nine samples yielded consistent PBMC viral load, differing by only 1.3-fold (range 1.0-1.7-fold). Among samples with detectable plasma HCV RNA, the correlation between PBMC and plasma viral load was moderate (r = 0.66) and was greater among HCV mono-infected compared to HIV/HCV co-infected subjects (r = 0.80 vs. 0.52). Measurement of cell-associated PBMC HCV RNA using a commercial assay demonstrated promising test characteristics. Differences in PBMC HCV viral load based on HIV-coinfection status and the significance of greater copy number in B-cells requires further study.

Persistent infection by HCV and EBV in peripheral blood mononuclear cells and risk of non-Hodgkin's lymphoma

Hepatitis C virus (HCV) and Epstein–Barr virus (EBV) have been repeatedly associated with risk of non-Hodgkin's lymphoma (NHL) in studies focusing on serological evidence of infection. We investigated NHL risk in association with detection of HCV-RNA or EBV-DNA in the peripheral blood mononuclear cells (PBMC). The study involved 91 NHL cases and 182 controls nested in the Italian branch of the EPIC (European Prospective Investigation of Cancer and nutrition) cohort, which obtained blood samples from 47,749 healthy volunteers between 1993 and 1998 in 5 Italian cities. NHL cases were identified until June 2005 through linkage with records of the Cancer, Mortality, and Hospital Discharge Registries. For all study subjects, we performed viral genome analyses on DNA and RNA extracted from buffy-coats and analysed EBV and HCV antibodies. The odds ratios (ORs) of NHL were 1.2 (95% confidence intervals: 0.4–3.8; 5 exposed cases) for PBMC HCV infection and 1.2 (0.7–2.3; 24 exposed cases) for PBMC EBV infection. Similar OR estimates were found for detection of EBV and HCV antibodies. These null results, although based on a relatively small sample size, suggest that persistent EBV and HCV infection in the PBMC is not a stronger predictor of NHL risk than serological evidence of infection.

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

We determined whether hepatitis C virus (HCV) RNA could be detected associated with peripheral blood mononuclear cells (PBMC) of seropositive blood donors who had spontaneously or therapeutically cleared their plasma viremia. Blood donor plasma viremia status was first determined with a highly sensitive transcription-mediated amplification (TMA) test performed in duplicate assays. PBMC from 69 aviremic and 56 viremic blood donors were then analyzed for the presence of HCV RNA with TMA adapted to detect viral RNA in PBMC and with a reverse transcription-nested polymerase chain reaction assay. PBMC-associated HCV RNA was detected in none of the 69 aviremic donors, including all 6 subjects with a sustained viral response following antiviral therapy. PBMC-associated HCV RNA was detected in 43 of the 56 viremic donors. The 13 viremic donors with no detectable PBMC-associated HCV RNA all had very low viral loads (6 positive only in 1 of 2 duplicate plasma TMA assays, 6 with viral loads below 100 HCV RNA copies/mL, and 1 with a viremia of 2700 HCV RNA copies/mL). The absence of detectable PBMC HCV RNA detection in all 69 aviremic donors reported here contrasts with prior studies, possibly as a result of the higher sensitivity of the TMA assay used to test for plasma viremia. Our results indicate that PBMC are unlikely to serve as a long-lived reservoir of HCV in aviremic subjects.

Compartmentalization of Hepatitis C Virus Genotypes between Plasma and Peripheral Blood Mononuclear Cells

Differences in hepatitis C virus (HCV) variants of the highly conserved 5' untranslated region (UTR) have been observed between plasma and peripheral blood mononuclear cells (PBMC). The prevalence and the mechanisms of this compartmentalization are unknown. Plasma and PBMC HCV variants were compared by single-strand conformation polymorphism (SSCP) and by cloning or by genotyping with a line probe assay (LiPA) in 116 chronically infected patients, including 44 liver transplant recipients. SSCP patterns differed between compartments in 43/109 analyzable patients (39%). Differences were significantly more frequent in patients with transplants (21/38 [55%] versus 22/71 [31%]; P < 0.01) and in those who acquired HCV through multiple transfusions before 1991 (15/20; 75%) or through drug injection (16/31; 52%) than in those infected through an unknown route (7/29; 24%) or through a single transfusion (5/29; 17%; P < 0.001). Cloning of the 5' UTR, LiPA analysis, and nonstructural region 5B sequencing revealed different genotypes in the two compartments from 10 patients (9%). In nine patients, the genotype detected in PBMC was not detected in plasma and was weak or undetectable in the liver in three cases. This genotypic compartmentalization persisted for years in three patients and after liver transplantation in two. The present study shows that a significant proportion of HCV-infected subjects harbor in their PBMC highly divergent variants which were likely acquired through superinfections.

Influence of Hemodialysis Procedure on HCV RNA Detection in Serum and Peripheral Blood Mononuclear Cells

Aim. To assess whether hemodialysis procedure induces qualitative or quantitative changes in hepatitis C virus (HCV) RNA. Methods. We obtained blood samples in the 10 HCV RNA‐positive patients of our hemodialysis unit before (sample I) and 5 min after a dialysis session (sample II), and before the next dialysis session (sample III). HCV RNA was tested by PCR in serum and peripheral blood mononuclear cells (PBMC). Serum viral load was measured by branched‐DNA assay. Results. Serum HCV RNA was positive in samples I, II and III of the 10 patients. PBMC HCV RNA was detected in samples I, II and III of seven patients. Mean viral load was 1.43 ± 0.99 Meq genome/mL in sample I, 0.86 ± 0.40 Meq genome/mL in sample II and 1.27 ± 0.56 Meq genome/mL in sample III. Conclusions. HCV load was low in most HCV RNA‐positive patients. It had a downward trend during dialysis procedure but HCV RNA remained detectable in all serum samples and in most PBMC samples. Therefore, qualitative HCV RNA seems to be better than viral load to assess HCV infection in hemodialysis patients.

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 10 7–10 8 copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10 9 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.

Differences in hypervariable region 1 quasispecies of hepatitis C virus in human serum, peripheral blood mononuclear cells, and liver.

Hepatitis C virus (HCV) has been reported to potentially replicate in peripheral blood mononuclear cells (PBMCs), but direct information on the pathogenic implication of HCV infection in PBMCs is still limited. To investigate this issue, we compared the complexity of HCV quasispecies in serum, PBMCs, and livers of 13 patients with type C chronic liver disease. Hypervariable region 1 (HVR 1) was amplified by reverse-transcription polymerase chain reaction (RT-PCR), and the PCR products were subcloned and sequenced. Considerable differences in the complexity of HVR 1 quasispecies were found in the serum, PBMCs, and liver in all patients, and the predominant sequences from each source were mutually different in 3 (23%) patients. An amino acid sequence unique to each source existed as well as a sequence common to serum and PBMCs, common to serum and livers, or common to PBMCs and liver. These results suggest infection of PBMCs by HCV, and that HCV in PBMCs may be differently exposed to host immunity from that in liver.

Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice.

The issue of infection of peripheral blood mononuclear cells (PBMC) by the hepatitis C virus (HCV) has potentially important implications, but is still debated. We have used the severe combined immunodeficiency (SCID) mouse model to test for the persistence of HCV in PBMC. Hematopoietic cells isolated from 14 subjects infected by HCV were inoculated intraperitoneally into SCID mice. Serum and blood cell samples from these mice were obtained with a mean follow-up of 8 weeks. As controls, human fibroblasts and sheep PBMC, preincubated with a human HCV-positive serum, were inoculated concomitantly into mice and analyzed. HCV-RNA positive strands were detected in 7 of 26 serum samples and 8 of 26 cell fractions from SCID mice inoculated with HCV-positive PBMC, after 8 weeks of follow-up. In contrast, no HCV RNA was detectable in the 10 control mice. HCV-RNA negative strands were detected in only 2 of 10 tested samples from 2 mice, and both positive mice had been inoculated with PBMC from HCV-positive subjects with malignant hematopoietic syndrome. Our study offers strong evidence for the persistence of HCV infection in mononuclear cells. Our results are also consistent with a low rate of HCV multiplication. This SCID mouse model might therefore be useful in analyzing the mechanisms of HCV persistence in mononuclear cells.

Hepatitis C virus RNA in plasma and blood mononuclear cells in patients with chronic hepatitis C treated with alpha‐interferon

Hepatitis C virus (HCV), a positive stranded RNA virus, is the main causative agent of post-transfusion and sporadic non-A non-B hepatitis worldwide. Paired samples of plasma and peripheral blood mononuclear cells (PBMC) from 11 patients with chronic hepatitis C treated with alpha-interferon (IFN) were tested, using a single step polymerase chain reaction (PCR), for the presence of HCV RNA. Before treatment, the viral genome was detected in all the plasma samples and 81.8% of PBMC. After 3 months of treatment, HCV RNA was still present in 63.6% of plasma samples but in only 27.3% of PBMC. A good correlation was observed between serum alanine aminotransferase level normalisation and disappearance of the viral genome in plasma. Among the six responder patients, five relapsed shortly after IFN withdrawal; HCV RNA became detectable again, especially in PBMC. These results show the presence of HCV in PBMC from most patients infected chronically. IFN therapy had an inhibitory effect on viral replication in lymphoid cells, but frequent relapses observed after cessation of treatment with IFN suggested persistence of HCV in these cells.

Testing for hepatitis C virus sequences in peripheral blood mononuclear cells of patients with chronic hepatitis C in the absence of serum hepatitis C virus RNA

Hepatitis C virus (HCV) is able to replicate in peripheral blood mononuclear cells (PBMC) of HCV‐infected patients. Few data are available on PBMC testing for HCV RNA in serum HCV RNA negative patients, positive for anti‐HCV and with histological evidence of chronic hepatitis. Twenty such patients were studied; of these, 11 were tested during interferon α (IFN) treatment, at the time of serum HCV RNA clearance and ALT normalisation: only one was found to be positive for HCV sequences in PBMC. Within 3 months of IFN withdrawal all 11 patients relapsed with high ALT and recurrence of serum HCV RNA. Of nine serum HCV RNA negative patients with chronic hepatitis C who were not receiving IFN when tested (four untreated patients and five patients who had already completed IFN schedule), PBMC HCV RNA was detected in four. Evidence of active HCV replication (presence of the minus strand genome) in PBMC was also observed in two cases. Thus, five of the 20 patients without detectable serum HCV RNA turned out to be carriers of HCV sequences in PBMC. These data indicate that: 1. PBMC are an extrahepatic replication site of HCV; this is true also in the absence of serum HCV RNA; 2. the role of PBMC as a “viral reservoir” after IFN‐induced serum HCV RNA clearance is questioned; 3. the absence of both serum and PBMC HCV RNA in patients under IFN is not predictive of sustained viral loss; 4. testing for PBMC viral sequences might enhance the chances of detecting HCV infection.

Detection of replicative form of hepatitis C virus RNA in peripheral blood mononuclear cells.

Paired samples of plasma and peripheral blood mononuclear cells (PBMC) from 7 patients with posttransfusion hepatitis C were collected and studied for the presence of replicative forms of hepatitis C virus (HCV). RNA was extracted and tested for both positive and negative strands of HCV RNA by polymerase chain reaction. Positive-strand RNA of HCV was found in both plasma and PBMC of all 7 patients. However, negative-strand RNA was found in PBMC of 3 patients while none was found in the plasma. Levels of negative-strand RNA were about 10-100 times less than those of positive-strand RNA by semiquantification with serial dilution of viral cDNA. The results suggest that active infection and replication of HCV in PBMC is present in patients with posttransfusion hepatitis C and implicate the extrahepatic infection of HCV.