Hepatitis C Virus RNA In Peripheral Blood Mononuclear Cells 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.

Seronegative occult hepatitis C infection among hemodialysis patients: A prevalence study.

The aim of this study was to investigate the prevalence of occult hepatitis C virus (HCV) infection (OCI) among HD patients. Blood samples were taken from 79 HD patients and their sera were evaluated for the presence of anti-HCV. Both the sera and peripheral blood mononuclear cells (PBMCs) were then checked for HCV RNA by nested reverse transcriptase-polymerase chain reaction. Anti-HCV was positive among 4/79 (5.1%) of the patients. From 75 patients who were negative for anti-HCV, 71 (94.7%) patients were also negative for HCV RNA in sera samples but five of them were positive for HCV RNA in PBMCs. Totally, out of 79 patients, HCV RNA was detected in PBMCs of five (6.3%) patients, indicating that these patients had OCI. No significant difference was observed between the frequency of OCI and gender (P-value = .6). HCV genotype in all five cases of OCI was genotype 3a. Our study showed prevalence rate of 6.3% OCI infection in HD patients. Regarding the serious complications and the clinical importance of OCI in HD patients, sensitive diagnostic methods for identifying HCV RNA in the PBMCs should be implemented for all HD patients.

Silent damage? Occult HCV replication and histological disease may occur following apparent HCV clearance

Silent damage? Occult HCV replication and histological disease may occur following apparent HCV clearance

Peripheral blood mononuclear cells hepatitis C virus RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients from the Damietta Governorate

Background Eradication of hepatitis C virus (HCV) infection is the goal of direct-acting antivirals with a high rate of sustained virological response (SVR). Currently, SVR is determined by negative serum HCV RNA by real-time (RT)-PCR that may not give any information about intracellular HCV replication. Aim To study peripheral blood mononuclear cells (PBMCs) HCV RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients. Patients and methods In all, 150 patients with chronic HCV, classified into 100 easy-to-treat patients (group I) and 50 difficult-to-treat patients (group II), who achieved SVR to sofosbuvir plus daclatasvir with or without ribavirin were enrolled in the study. HCV RNA was evaluated in both sera and isolated PBMCs using the polymerase chain at 3 and 12 months from the end of treatment (EOT). Results As regards HCV RNA in peripheral blood mononuclear cells (PBMCs) we found that no case was positive in the easy to treat (group I). In difficult to treat (group II), six (4%) patients were positive at 3 months and 24 (16%) patients at 12 months after EOT. However, there is no virological, clinical, or biochemical relapse noted during the follow-up period among positive cases. All positive cases were cirrhotic, with significantly lower platelets count and albumen level but higher bilirubin than group I. Conclusion All easy-to-treat groups were HCV RNA in PBMCs negative at EOT and during the follow-up period (1 year). Follow-up of cirrhotic patients with positive HCV RNA in PBMCs showed no clinical, biochemical, or virological relapse.

HCV Therapy Follow-up Fractionation (CTF2) by Intra-PBMC Nested RNA PCR Recognizes Early Virologic Response and Relapse.

Background and Aims: Sustained virologic response is evaluated by single-step reverse transcription (SRT) PCR assay, which assesses hepatitis C virus (HCV) clearance from plasma but not from tissues such as peripheral blood mononuclear cells (PBMCs). Persistence of HCV RNA in PBMCs beyond end of treatment (EOT) is associated with nonresponse. Our goal was to measure intra-PBMC HCV RNA levels during oral antiviral therapy according to the HCV therapy follow-up fractionation (CTF2) protocol.Methods: Compensated chronic HCV patients (n = 2 78 SRT-PCR positive) were scheduled to receive oral antiviral therapy. Subjects were followed-up by SRT and intra-PBMCs HCV RNA PCR at the end of the 2nd, 6th, 10th, 14th, 18th and 24th weeks to evaluate virus clearance from plasma and PBMCs, respectively. The CTF2 protocol evaluated SRT and PBMC PCR status at each follow-up point for determining therapy continuation or interruption to address cost effectiveness.Results: All patients tested negative by SRT PCR after therapy for 2 weeks. Application of the CTF2 protocol revealed: a) increasing HCV clearance rate from 75.9% at the end of 10th week to 90.3% at the end of 24th week (p < 0.00001); b) faster clearance of HCV from plasma compared to PBMCs at each point of follow-up until the 18th week (p < 0.05); c) higher viral elimination rates diagnosed by PBMC HCV RNA PCR(−) compared to PBMC HCV RNA PCR(+) from the 6th to 24th week of treatment (p < 0.0001); d) higher over-time increase curve of combined plasma and PBMC HCV RNA determined negativity compared to the decline in positivity curves by PBMC PCR at the 6th-18th week compared to the 24th week (p < 0.01)—these results validated treatment continuation; and e) solitary evaluation of EOT sustained HCV infection and relapses by PBMC HCV RNA (p < 0.001).Conclusions: Early elimination of serum and tissue (PBMC) HCV infection by oral antiviral therapy can be achieved and evaluated during a cost-effective CTF2 protocol application.

The Absence of HCV RNA and NS5A Protein in Peripheral Blood Mononuclear Cells Is a Prognostic Tool for Sustained Virological Response.

Hepatitis C Virus (HCV) infection is a major health concern worldwide. The presence of both HCV viral RNA and NS5A proteins in peripheral blood mononuclear cells (PBMCs) indicate the efficacy of the treatment during sustained virological response (SVR) and end of treatment response (ETR). The main objective of this study was to detect the absence or presence of HCV RNA and NS5A proteins in PBMCs. Blood samples were taken from selected patients (Islamabad, Pakistan) before treatment, at ETR, and during SVR. Two hundred HCV responders to pegylated IFN-α-2a plus ribavirin were selected. HCV RNA was extracted from the patients to determine the viral load by reverse transcription (RT)-polymerase chain reaction before treatment. Out of 200 patients, 152 (76%) and 48 (24%) achieved positive and negative ETR, respectively. Among ETR patients, 134 (88.2%) showed SVR, whereas 18 (11.8%) displayed relapse. The male to female ratio was 92:108 with mean age of 37.4 years. Among 152 ETR-positive patients, 29 (19%) patients' PBMCs were positive for HCV RNA and 27 (17.8%) were positive for NS55A proteins. Patients having HCV RNA in PBMCs showed higher relapse frequency compared with patients lacking it. Similarly, patients having NS5A protein showed significantly higher relapse frequency compared with patients lacking it. All PBMC-positive samples were of genotype 3a. In addition, patients with positive NS5A in their PBMCs showed greater risk of relapse compared with patients having HCV RNA. We conclude that the absence of both viral HCV and proteins can be used as an indicator for diagnosis of SVR in the future.

Assessment of Hepatitis C Virus RNA in Peripheral Blood Mononuclear Cells as a Predictor of Response to Pegylated-Interferon and Ribavirin: A Cohort Study

Background: Sustained virologic response (SVR) to pegylated-interferon (PegIFN) and ribavirin (RBV) in hepatitis C virus (HCV)-infected patients could be predicted by detection of serum HCV RNA whereas detection of HCV RNA in other reservoirs such as peripheral blood mononuclear cells (PBMCs) for prediction of treatment response is still a mystery. Objectives: This study aimed at assessing the prediction of SVR by detection of HCV RNA in PBMCs or serum in patients during treatment. Methods: In a cohort study (2011 to 2014), 100 chronic HCV patients at Tehran Hepatitis Center were treated with PegIFN and RBV. Serum HCV RNA level was measured at baseline, 4, 12, and 24 weeks during treatment and at 24 weeks after termination of treatment. Meanwhile, HCV RNA was evaluated in PBMCs at weeks 4, 12, and 24 during the treatment. Results: Out of 100 patients treated in this study, 91 completed the course of treatment. Most patients were young males infected with HCV genotype 1. Cirrhosis and previous history of treatment was found in 16.5% and 26.5% of patients. Sustained virologic response was achieved in 65 (71.4%) patients. Among baseline parameters, only female gender was significantly associated with SVR. Undetectable serum HCV RNA at week 4 (OR = 4.74) and week 12 (OR = 11.63) of treatment predicted SVR rate while the same was not true for detection of serum HCV RNA at week 24 of treatment. Moreover, detection of HCV RNA in PBMCs at weeks 4 and 12 of treatment was not associated with the rate of SVR, while absence of HCV RNA in PBMCs at week 24 of treatment was associated with SVR (OR = 4.55). Conclusions: Detection of HCV RNA in PBMCs, especially at week 24 of treatment with PegIFN and RBV, could be considered as an additional marker for prediction of treatment response. It is recommended to assess HCV on-treatment kinetic in PBMCs of patients treated with direct-acting antiviral agents for prediction of treatment response.

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

Insights into the role of HCV Plus-/Minus strand RNA, IFN-γ and IL-29 in relapse outcome in patients infected with HCV.

Approximately one-third of hepatitis C virus (HCV) infected patients who complete antiviral therapy with undetectable serum HCV RNA at the end of therapy (ETR), will experience relapse. The reasons for the failure of treatment have not been elucidated. It was showed that HCV RNA can persist and replicate in extra hepatic sites, e.g. in peripheral blood mononuclear cells (PBMCs), but the relevance of its presence with relapse over time is still unknown. Moreover, interferon-gamma (IFN-γ) and IFN-lambdas [IFN-λ1, interleukin-29 (IL-29)], possess potent antiviral activity. We studied if the presence of plus-/minus strand RNA in PBMCs of patients and the serum level of IFN-γ and IL-29, which is the most abundant IFN-lambdas in serum, can be considered as predictive factors in relapse outcomes. Patients were screened for plus-/minus strand RNA at ETR and after 6 months. Also, we measured the serum level of IFN-γ and IL-29 and compared the result with those who developed a sustained virological response (SVR). Levels of IL-29 and IFN-? serum were significantly higher in SVR at ETR and 6 months later compared to those of the relapsed patients, but there was no difference between the two groups regarding the presence or absence of plus-/minus HCV strand in PBMCs. Our novel findings showed that the serum level of IL-29 and IFN-γ are predictive of relapse outcomes to HCV treatment, but there was no association between the presence of plus-γminus HCV RNA in PBMCs of patients with an outcome of therapy at ETR and later.

Long-term follow-up of successful hepatitis C virus therapy: waning immune responses and disappearance of liver disease are consistent with cure.

A sustained viral response (SVR) after interferon-based therapy of chronic hepatitis C virus (HCV) infection is regarded to represent a cure. Previous studies have used different markers to clarify whether an SVR truly represents a cure, but no study has combined a clinical work-up with highly sensitive HCV RNA detection, and the determination of immune responses. To determine clinical, histological, virological and immunological markers 5-20 years after SVR. In 54 patients, liver biochemistry, histology and elastography were evaluated. Liver biopsies, plasma and peripheral blood mononuclear cells (PBMCs) were tested for minute amounts of HCV RNA. HCV-specific T-cell responses were monitored by ELISpot and pentamer staining, and humoral responses by measuring HCV nonstructural (NS)3-specific antibodies and virus neutralisation. Liver disease regressed significantly in all patients, and 51 were HCV RNA-negative in all tissues tested. There was an inverse association between liver disease, HCV-specific T-cell responses and HCV antibody levels with time from SVR, supporting that the virus had been cleared. The three patients, who all lacked signs of liver disease, had HCV RNA in PBMCs 5-9 years after SVR. All three had HCV-specific T cells and NS3 antibodies, but no cross-neutralising antibodies. Our combined data confirm that a SVR corresponds to a long-term clinical cure. The waning immune responses support the disappearance of the antigenic stimulus. Transient HCV RNA traces may be detected in some patients up to 9 years after SVR, but no marker associates this with an increased risk for liver disease.

Genetic variations in host IL28B links to the detection of peripheral blood mononuclear cells-associated hepatitis C virus RNA in chronically infected patients

Hepatitis C virus (HCV) is mainly hepatotropic; however, several reports document the presence of genomic viral RNA in extrahepatic sites including peripheral blood mononuclear cells (PBMCs). In this study, the presence of HCV RNA was initially evaluated in the plasma and peripheral blood mononuclear cells (PBMCs) of 53 HCV-infected patients who were treated per protocol. PBMC-associated HCV RNA was detectable in 79% of patients. Early virological response to combined pegylated interferon-α (PegIFN) and ribavirin (RBV) therapy in patients with undetectable levels of PBMCs-associated HCV RNA was 100%, while it was 60% (P = 0.003) in those who had detectable levels of PBMC-associated HCV RNA. A sustained virological response was observed in 35% of patients with detectable PBMC-associated HCV RNA, but was 70% in patients with undetectable levels of PBMC-associated HCV RNA (P = 0.07). In a multivariate analysis incorporating parameters such as HCV genotype, viral load, presence of cirrhosis and absence of PBMC-associated HCV RNA, a significant relationship was observed between the detection of PBMC-associated HCV RNA and the sustained virological response (OR 19.4, 95% CI: 2.1-486.2, P = 0.0061). The association between single nucleotide polymorphism (SNP) in IL28B, known predictor of antiviral therapy outcome, and the occurrence of HCV RNA in PBMC in 84 chronically infected patients was then evaluated. Results suggest that the presence of a G allele in rs8099917, known to associate to a poor response to PegIFN/RBV therapy, also predicts an increased association of HCV RNA with PBMC (OR: 3.564; 95% CI: 1.114-11.40, P = 0.0437).

Evaluation of Hepatitis C Virus Infection in Antibody Positive Orphan Newborns

Background: The rate of mother-to-infant hepatitis C virus (HCV) transmission is relatively low, however, this mode of infection is the most important route for childhood HCV involvement. The chances of transmission are increased by the level of maternal HCV viraemia and maternal human immunodeficiency virus (HIV) co infection. Objectives: The objective of the present study was to determine the presence of HCV infection in orphan newborns of infected mothers. Patients and Methods: A total of 29, two to seven month old, orphan infants were included in the study. The tests performed consisted of the detection of; anti-HCV and anti-HIV antibodies, HCV RNA in the plasma and peripheral blood mononuclear cells by real-time polymerase chain reaction (PCR), and finally the detection of HCV core antigens (HCV core Ag). Results: Anti-HCV antibodies were detectable in all of the infants. However HCV RNA was undetectable in both plasma and peripheral blood mononuclear cells (PBMCs) and there were also no anti-HIV 1/2 antibodies present in the infants. Conclusions: Few studies have addressed the issue of mother-to-infant transmission of HCV among orphan newborns. According to the present study, carried out with orphan babies, the use of a HCV core Ag assay and evaluation of HCV RNA in PBMCs together with HCV RNA real-time PCR on the plasma, could benefit the prediction or exclusion of HCV transmission from mother to child.

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.

Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C.

To investigate the existence and clinical significance of hepatitis C virus (HCV) RNA in the serum and peripheral blood mononuclear cells (PBMC) of patients with hepatitis C. HCV RNA was detected by nested polymerase chain reaction (Nested PCR) in serum and in PBMC of 46 patients with acute hepatitis C (AHC) and in 42 patients with chronic hepatitis C (CHC). The positive rate of HCV RNA in PBMC of patients with CHC was markedly higher than that of patients with AHC (P < 0.01). The positive rates of HCV RNA in serum of patients with AHC and CHC and in PBMC of patients with CHC were significantly higher than those of anti-HCV positive patients with normal alanine aminotransferase (ALT) levels (P < 0.01). HCV RNA was negative in the serum of two patients, but could be detected in PBMC. In 12 patients, anti HCV was negative while HCV RNA was positive in serum. (1) detection of serum HCV RNA by nested PCR might be helpful in the early diagnosis of anti-HCV negative hepatitis C; (2) liver damage in patients with hepatitis C might be correlated with HCV-viremia; (3) infection of PBMC by HCV might play an important role in chronic liver damage in patients with HCV and in the chronicity of its clinical course; and (4) PBMC might be considered as a "reservoir" for HCV.

Clearance of hepatitis C virus RNA from serum in HIV/hepatitis C virus coinfection indicates eradication from peripheral blood mononuclear cells

The objectives of this study are to determine the frequency of hepatitis C virus (HCV) RNA persistence in peripheral blood mononuclear cells (PBMCs) of HIV-positive patients with clearance of the virus from serum and to identify the presence of any ongoing replication. This is a prospective cross-sectional study. HIV antibody-positive individuals with previous exposure to HCV, but not current infection with HCV, were recruited. Blood was taken to allow identification of HCV RNA in both serum and PBMCs. Intracellular HCV was extracted using the QIAamp RNA Blood MiniKit. Reverse transcriptase-PCR was performed using a modification of the COBAS TaqMan HCV Test for use with the high pure system. Twenty-six HIV-positive individuals were recruited to the study. All had previously been infected with HCV. Six individuals had spontaneously cleared HCV, 10 had achieved sustained virological response following 24 weeks of pegylated interferon and ribavirin for acute HCV, and 10 had achieved sustained virological response following standard pegylated interferon and ribavirin therapy for chronic HCV. None demonstrated HCV RNA persistence in either serum or PBMCs. Our findings lend support to the view that clearance of HCV RNA from serum in HIV/HCV coinfection indicates eradication from PBMCs. Thus, absence of serum HCV RNA 6 months after the end of therapy can be used as a marker of treatment success for interferon-based therapies. However, the advent of small molecule HCV inhibitors may require us to rethink our definitions of response and cure.

Expression of microRNA-155 precursor in peripheral blood mononuclear cells from hepatitis C patients after antiviral treatment

Chronic hepatitis caused by Hepatitis C virus (HCV) is the main source of liver cirrhosis, hepatocellular carcinoma, and extra-hepatic diseases. After treatment-induced resolution of hepatitis C, the persistence of HCV RNA in serum and peripheral blood mononuclear cells (PBMCs) is often observed. An expression of the precursor of microRNA-155 (miR-155) called BIC can be the factor responsible for a course of HCV infection. Therefore, we assessed the relationship between BIC expression and HCV RNA status in sera and PBMCs samples of 64 hepatitis C patients treated with interferon alpha(IFN-alpha)+ribavirin. High expression of BIC in PBMCs was determined in 100% of patients that harbored HCV RNA in serum and PBMCs. Further, we found that 83% of PBMCs samples were BIC-positive in a group of patients that eliminated HCV RNA only from serum. The lowest expression of BIC was found in patients that eliminated HCV RNA from both serum and PBMCs.

Rapid virological response in peripheral blood mononuclear cells with an increase of hepatitis C virus-specific interferon-gamma production predisposes to sustained virological response in patients with chronic hepatitis C genotype 1 undergoing treatment with pegylated-interferon alpha 2a plus ribavirin

Objective. Viral load evaluation in plasma, after 1 month of treatment, represents one of the most important parameters to predict treatment response during interferon (IFN) treatment in chronic hepatitis C (CHC). It has been proven that hepatitis C virus (HCV) RNA may be present in peripheral blood mononuclear cells (PBMCs) but few studies have investigated the viral load in PBMCs during treatment. The aim of this study was to evaluate HCV RNA in PBMCs during therapy with pegylated-IFN-α2a plus ribavirin and whether its clearance in PBMCs may induce a treatment response. Furthermore, we also analyzed the IFN-γ and interleukin (IL)-4 responses of PBMCs during therapy. Material and methods. We studied 35 patients with CHC genotype 1 undergoing antiviral treatment with pegylated IFN-α2a 180 μg weekly plus ribavirin 1000 mg/daily. In these patients we evaluated HCV-RNA in plasma and PBMCs, IFN-γ and IL-4 before treatment, after 1, 3 and 12 months of treatment and 6 months after the end of treatment. Results. We found that rapid virological clearance of HCV-RNA in PBMCs with a restored and improved HCV-specific IFN-γ response was statistically significantly higher in those with a sustained virological response (SVR). Conclusion. Patients having a rapid virological response in PBMCs with an improved Th1 network achieve a complete SVR, whereas those having viral clearance only in plasma without a restored Th1 network have a relapse.

The myth of occult hepatitis C infection

We read with interest the review by Welker and Zeuzem on occult hepatitis C virus (HCV) infection and also the reply by Carreno et al.1, 2 The latter reported in their answer to Welker and Zeuzem that HCV can replicate in patients with occult HCV infection. Because this conclusion would have substantial consequences for patients considered cured of their HCV infection, as well as for the health care system, the data must be interpreted most carefully. They reported two studies that “unconvincingly failed to detect occult HCV infection in cryptogenic hepatitis because of several concerns with the methodology employed.”3, 4 We would like to correct Carreno et al. on these two important points. In the first study published by Tanari et al., it was shown that the ability for HCV RNA detection is more sensitive in whole blood instead of peripheral blood mononuclear cells (PBMCs), due to circulation of the infectious virus in patient sera in the form of triglyceride-rich particles which is present in the whole blood and not in PBMCs.5 In the second study, we looked for the so-called “occult hepatitis C”, in a different population of patients and controls, including patients with long-term normal alanine aminotransferase level, by using an original ultrasensitive validated real-time polymerase chain reaction (PCR) method. We failed to find any occult hepatitis C.4 Moreover, we would like to underscore that for a long time, Carreno et al. reported the same results on the same cohort of patients.6 The method proposed used PBMCs from a patient chronically infected with HCV for RNA isolation and HCV RNA detection. Serial dilutions of the isolated RNA cannot be a gold standard due to the limit of detection of HCV RNA by an in-house reverse transcription PCR method. PBMCs from our patients were subjected to HCV RNA transcription-mediated amplification assays without any HCV RNA detectable using this high sensitivity assay.6 The second issue addressed is related to the concept of the presence of HCV RNA in the liver in the absence of HCV RNA detected in PBMCs. Carreno et al. suggested that negativity for HCV-RNA in PBMC does not exclude the existence of occult HCV infection because the gold standard method to identify this occult infection is by detection of viral RNA in liver cells. To address this question, Maylin et al. recently assessed the presence of residual HCV RNA in serum, liver, and PBMCs using TMA (sensitivity <9.6 IU/mL) in a long-term follow-up study of patients infected with chronic hepatitis C who achieved a sustained virological response after interferon-based treatment.6 A total of 114 patients had a posttreatment liver tissue and 156 had PBMCs. Serum HCV RNA remained undetectable (1300 samples), indicating that none of the patients had a relapse. HCV RNA was detectable in 2 of 114 (1.7%) liver specimens, and in none of 156 PBMC specimens. These authors strongly suggest that SVR may be considered to show eradication of HCV infection.7 This latest observation was confirmed by another recent study with a complete clearance of HCV RNA from the PBMCs of blood donors who spontaneously or therapeutically control their plasma viremia.8 In conclusion, the excellent review performed by Welker and Zeuzem on occult HCV infection confirm our data and those of two other studies using sensitive assays and liver/PBMC assessment, and reinforce the absence of evidence of occult HCV infection. Philippe Halfon M.D., Ph.D.*, Michele Martinot-Peignoux Ph.D. , Patrice Cacoub M.D., Ph.D. , * Département Virologie Laboratoire Alphabio, Service Maladie Infectieuses Hôpital Ambroise Paré, Marseille, France, Université Paris VII, Hôpital Beaujon, Clichy, France; Service de Microbiologie, Hôpital Beaujon, Clichy, France; Institut National de la Santé et de la Recherche Médicale U-773, Centre de Recherche Biomédicale Bichat-Beaujon CRB3, Hôpital Beaujon, Clichy, France, Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique, UMR 7087, Paris, France; Hôpital Pitié-Salpêtrière, Service de Médecine Interne, Assistance Publique–Hôpitaux de Paris, Paris, France.

Occult hepatitis C: How convincing are the current data?

Occult hepatitis C: How convincing are the current data?

Persistence of Hepatitis C Virus in Peripheral Blood Mononuclear Cells of Sustained Viral Responders to Pegylated Interferon and Ribavirin Therapy

The aim of this paper was to assess the persistence of hepatitis C virus (HCV) among patients successfully treated with peginterferon and ribavirin. The persistence of viral RNA was evaluated in the serum and peripheral blood mononuclear cells (PBMCs) of 25 chronic hepatitis C patients with sustained viral response to peginterferon and ribavirin treatment up to 56 months after the completion of therapy. Viral RNA was detected in the peripheral blood mononuclear cell cultures of five patients (20%), but none had detectable serum HCV RNA. At present, the clinical relevance of this finding is unclear. It is possible that viral persistence and, specifically, the presence of HCV RNA in PBMCs may lead to HCV reactivation under special circumstances, such as immunosuppression.