Liver Hepatitis C Virus RNA Research Articles

Sofosbuvir/Velpatasvir Prophylaxis for 12 Weeks in Hepatitis C Virus (HCV)-Negative Recipients Receiving Kidney Transplantation from HCV-Positive Donors.

BackgroundThe aim of this study was to evaluate the efficacy and safety of Sofosbuvir/Velpatasvir prophylaxis in hepatitis C virus (HCV)-negative recipients who received a transplant kidney from HCV-infected donors.Material/MethodsThis retrospective cohort study enrolled consecutive HCV-negative recipients between January 2019 and February 2021. All the recipients were treated with Sofosbuvir/Velpatasvir (400 mg/100 mg) once daily for 12 weeks after receiving a transplant kidney from HCV-infected donors. We collected data on renal function and liver function and HCV RNA were collected during the study. We also compared the rates of adverse events.ResultsA total of 26 patients were included in the cohort. All the recipients (100%) completed 12 weeks of treatment and the entire follow-up. All recipients (100%) had negative HCV RNA, but 4 recipients (15.4%) were HCV antibody (Ab)-positive after transplantation. Fifteen adverse events (57.7%) occurred during the study. Three recipients (11.5%) experienced graft rejection, 6 recipients (23.1%) had delayed graft function, and 3 recipients (11.5%) had bleeding. However, none of them were related to study medication. Renal function was stable in all patients.ConclusionsSofosbuvir/Velpatasvir pre- and post-transplantation treatment was effective and safe in HCV-uninfected recipients who received a transplant kidney from HCV-infected donors.

MEASUREMENT OF SERUM BIOCHEMICAL LIVER PARAMETERS AND HCV RNA LEVELS FOR DETECTION THE SEVERITY OF HEPATITIS C VIRUS-ASSOCIATED HEPATOCELLULAR CARCINOMA

Hepatitis C virus (HCV) is one of the leading causes of chronic liver disease. Hepatocellular carcinoma (HCC) is a major complication associated with HCV virus infection, with significant mortality and morbidity rates. This study aimed to measure biochemical liver parameters and HCV RNA levels for detection the severity of hepatitis C virus- associated hepatocellular carcinoma. The study was conducted on 100 patients, with ages ranging from 36 to 68 years and patients grouped into four groups. The 1 st group served as control group (n= 25),the second group (n=25): Hepatitis C Virus, the third group (n=25): HCV-associated HCC and the fourth group (n=25): After HCC removal and tumor resection. Serum samples were collected from the studied patients. Liver function enzymes (ALT, AST, Alkaline phosphatase) and another function parameters (Albumin and Total bilirubin) were tested to all patients of the studied groups. The results showed that hepatitis C Virus, HCV-associated HCC, and after HCC removal groups had an increase in liver function enzymes, decrease in albumin levels, and an increase in total bilirubin levels which indicate damage in the liver. Viral loads indicated in males infected higher than in females and significantly increased in HCV patients, and a highly significant increase in HCV associated HCC patients. Conclusively, Hepatitis C Virus, HCV-associated HCC, and After HCC removal groups had an increase in liver function enzymes, decrease in albumin levels, and an increase in total bilirubin levels which indicate damage in the liver. Viral loads indicated in males infected higher than females are significantly increased in HCV patients, and a highly significant increase in HCV associated HCC patients.

Prediction of HCV load using genotype, liver biomarkers, and clinical symptoms by a mathematical model in patients with HCV infection.

Hepatitis C virus (HCV) infection is a major public health problem with about 1.75 million new HCV cases and 71 million chronic HCV infections worldwide. The study aimed to evaluate clinical, serological, molecular, and liver markers to develop a mathematical predictive model for the quantification of the HCV viral load in chronic HCV infected patients. In this cross-sectional study, blood samples were taken from 249 recently diagnosed HCV-infected subjects and were tested for liver condition, viral genotype, and HCV RNA load. Receiver operating characteristics (ROC) curves and multiple linear regression analysis were used to predict the HCV-RNA load. Genotype 3 followed by genotype 1 were the most prevalent genotypes in Mashhad, Northeastern Iran. The maximum levels of viral load were detected in the mixed genotype group, and the lowest levels in the undetectable genotype group. The log of the HCV viral load was significantly associated with thrombocytopenia and higher serum levels of alanine transaminase (ALT). In addition, the log HCV RNA was significantly higher in patients with arthralgia, fatigue, fever, vomiting, or dizziness. Moreover, genotype 3 was significantly associated with icterus. A ROC curve analysis revealed that the best cut-off points for serum levels of aspartate aminotransferase (AST), ALT, and alkaline phosphatase (ALP) were >31, >34, and ≤246 IU/L, respectively. Sensitivity, specificity, and positive predictive values for AST were 87.7%, 84.36%, and 44.6%, for ALT they were 83.51%, 81.11%, and 36%, and for ALP were 72.06%, 42.81%, and 8.3%, respectively. A mathematical regression model was developed that could estimate the HCV-RNA load. Regression model: log viral load = 7.69 - 1.01 × G3 - 0.7 × G1 + 0.002 × ALT - 0.86 × fatigue.

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

Evaluation of Serum Alpha-Fetoprotein Level in Chronic Hepatitis C Patients.

Hepatitis C virus (HCV) infection represents an important risk factor for hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is a widely used biomarker for HCC. However, elevated serum AFP levels in different statuses of chronic hepatitis C (CHC) is not well defined. This study aimed to evaluate the relationship between AFP levels and HCV viral load in CHC patients. We also analyzed the correlation between three liver func-tion enzyme levels (AST, ALT, GGT) and HCV RNA viral load in CHC patients. A total of 279 patients infected with HCV were included in this study. Patients were divided into two groups: HCV RNA positive and HCV RNA negative group. Serum HCV RNA load was measured by Quantitative Real-time PCR. Electrochemiluminescence assay (ECLA) was used to determine the serum AFP levels. The differences between two groups in AFP levels and biochemical profile (AST, ALT, GGT) was evaluated. The HCV RNA-positive group had significantly higher serum AFP levels than the negative groups (12.61 vs. 4.72 ng/mL, p < 0.0001). There was no correlation between AFP levels and HCV RNA viral load in HCV infection patients (p = 0.92). A significant correlation was observed between serum ALT (r = 0.243, p = 0.005), GGT (r = 0.212, p = 0.016) levels and HCV RNA viral load. Poor correlation (r = 0.148, p = 0.093) was found between AST levels and HCV RNA viral load. Interestingly, there was a positive correlation (r = 0.337, p < 0.001) between ALT and AFP levels in the whole study population. We concluded that serum HCV RNA positive patients were candidates for therapeutic prevention of HCC and liver inflammation regardless of the HCV RNA viral load. Furthermore, higher burden of HCV viral load was associated with more severe liver damage.

Liver enzyme normalization predicts success of Hepatitis C oral direct-acting antiviral treatment.

Monitoring of hepatitis C virus (HCV) treatment response is performed by serial HCV RNA measurements; however, this may not be useful for predicting treatment success or failure with oral direct-acting antiviral agent (DAA) therapies. Liver enzyme levels, which are elevated in chronic HCV and tend to decline on therapy, may serve as a more logistically and economically feasible alternative for monitoring treatment response. The Ottawa Hospital Viral Hepatitis Clinic patients (n=219), receiving interferon-free oral DAA treatments, were assessed for liver enzymes and HCV RNA levels at baseline, week 4 and ≥12 weeks post-treatment. Suppression cut points used for this analysis were ALT ≤ 40U L-1 and AST ≤ 30U L-1. The primary outcome was week 12 sustained virologic response (SVR). By our analysis, all indicators had strong PPV (>90%) but limited NPV (.

Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy.

Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.

Hepatitis C virus reactivation in cancer patients in the era of targeted therapies.

The purpose of this review is to summarize the evidence of hepatitis C reactivation in cancer patients in the era of targeted therapies. Targeted therapies are novel therapeutics frequently used in cancer patients. During treatment with targeted therapies, viral replication is one of the major problems that can occur. The PubMed database, ASCO, and ASCO Gastrointestinal Cancer Symposium abstracts were searched up until September 15, 2013 using the following search keywords: "targeted therapies, rituximab, alemtuzumab, brentuximab, hepatitis, hepatitis C reactivation, tyrosine kinase inhibitors, imatinib, mammalian target of rapamycin (mTOR) inhibitors, everolimus, anti-HER therapies, trastuzumab, pertuzumab, lapatinib, anti-epidermal growth factor receptor therapies, cetuximab, panitumumab, and ipilimumab". Papers considered relevant for the aim of this review were selected by the authors. The data about rituximab-induced hepatic flare in hepatitis C virus (HCV) positive patients is controversial. However, there is the possibility of life-threatening hepatic flare that can develop after HCV ribonucleic acid (HCV-RNA) viral load increases. Routine follow-up of liver function tests should be advised. Especially in high-risk patients, such as those with baseline chronic active hepatitis and cirrhosis, and where there are plans to administer rituximab concomitantly with corticosteroids, it is advised to have close follow-up of HCV viral load. The data is insufficient to make accurate statements about the association of alemtuzumab therapy and HCV reactivation. However, alemtuzumab may cause deep immunosuppression. Due to this, it is better to follow up with liver function tests and HCV RNA levels during alemtuzumab therapy. Brentuximab has effects on antibody dependent cellular toxicity and may decrease humoral immunity. Thus, we believe that during brentuximab treatment of HCV infected patients, clinicians may encounter hepatitis C reactivation. There have been no reported cases of hepatitis C reactivation with imatinib therapy. However, there are many reports of hepatitis B reactivation with imatinib treatment. Based on the evidence of hepatitis B reactivation with imatinib and the effects of imatinib on immune system functions, we suggest that imatinib therapy might be a risk factor for HCV reactivation. Anti-human epidermal growth factor receptor 2 therapies are not associated with hepatic flare in HCV infected patients. Post-transplant studies reported that mTOR was safely administered to patients with active hepatitis C without causing hepatic flare. Cetuximab and panitumumab have not been associated with HCV reactivation. Two cases of HCV infected melanoma were safely treated with ipilimumab without any HCV reactivation or hepatic flare. Targeted therapies are a new and emerging area of oncology treatment modalities. While treating HCV infected cancer patients, clinicians should be mindful of the immunosuppressive properties of targeted therapies. Further randomized trials are needed to establish algorithms for this issue.

Interferon-λ4 (IFNL4) Transcript Expression in Human Liver Tissue Samples

Eradication of hepatitis C virus (HCV) infection, both spontaneous and treatment-induced, is marked by the wildtype allele C of a single nucleotide polymorphism upstream of the IL28B gene, rs12979860. This favorable allele was recently described to be in linkage disequilibrium with the wildtype allele TT of a dinucleotide polymorphism, ss469415590, located within a new protein-coding gene. While the TT allele introduces a frame-shift and disrupts the open reading frame, only the variant allele, ΔG, creates a novel type III interferon (IFN) protein, IFN-λ4/IFNL4. Absence of IFNL4 is thus supposed to favor resolution of HCV infection. As to date IFNL4 mRNA transcription has only been investigated in polyI:C-stimulated primary human hepatocytes and not yet in HCV infection in vivo, this study analyzed IFNL4 mRNA expression in human liver biopsy specimens. Samples were obtained from patients with a broad panel of disorders including no liver disease, liver diseases of non-viral etiology, chronic hepatitis B and chronic hepatitis C. Hepatic IFNL4 transcripts were detectable exclusively in a subgroup of chronic hepatitis C patients (24/45). Their amounts were positively related to liver HCV RNA copy numbers (p = 0.0023, r = 0.56) suggesting that the hepatic viral load influences IFNL4 transcription irrespective of IFNL4 governing genotype. Both, the IFNL4 creating allele ΔG (p<0.0001) and actual IFNL4 transcription (p = 0.0015) were found to be correlated to the activation of IFN stimulatory genes (ISGs). By contrast, IFNL4 ss469415590 genotypes were not found to be related to IFN-λ2/3/IL28 or IFN-λ1/IL29 gene expression. In conclusion, this study is the first report on intrahepatic transcript levels of the recently discovered IFNL4 gene. Data indicate that HCV infection in particular might activate IFNL4 transcription in the liver. It provides a possible explanation as to why hepatitis C patients show ISG stimulation in their livers in the apparent absence of an induction of other IFN subtypes.

New perspectives in occult hepatitis C virus infection

Occult hepatitis C virus (HCV) infection, defined as the presence of HCV RNA in liver and in peripheral blood mononuclear cells (PBMCs) in the absence of detectable viral RNA in serum by standard assays, can be found in anti-HCV positive patients with normal serum levels of liver enzymes and in anti-HCV negative patients with persistently elevated liver enzymes of unknown etiology. Occult HCV infection is distributed worldwide and all HCV genotypes seem to be involved in this infection. Occult hepatitis C has been found not only in anti-HCV positive subjects with normal values of liver enzymes or in chronic hepatitis of unknown origin but also in several groups at risk for HCV infection such as hemodialysis patients or family members of patients with occult HCV. This occult infection has been reported also in healthy populations without evidence of liver disease. Occult HCV infection seems to be less aggressive than chronic hepatitis C although patients affected by occult HCV may develop liver cirrhosis and even hepatocellular carcinoma. Thus, anti-HCV negative patients with occult HCV may benefit from antiviral therapy with pegylated-interferon plus ribavirin. The persistence of very low levels of HCV RNA in serum and in PBMCs, along with the maintenance of specific T-cell responses against HCV-antigens observed during a long-term follow-up of patients with occult hepatitis C, indicate that occult HCV is a persistent infection that is not spontaneously eradicated. This is an updated report on diagnosis, epidemiology and clinical implications of occult HCV with special emphasis on anti-HCV negative cases.

Human cell types important for Hepatitis C Virus replication in vivo and in vitro. Old assertions and current evidence

Hepatitis C Virus (HCV) is a single stranded RNA virus which produces negative strand RNA as a replicative intermediate. We analyzed 75 RT-PCR studies that tested for negative strand HCV RNA in liver and other human tissues. 85% of the studies that investigated extrahepatic replication of HCV found one or more samples positive for replicative RNA. Studies using in situ hybridization, immunofluorescence, immunohistochemistry, and quasispecies analysis also demonstrated the presence of replicating HCV in various extrahepatic human tissues, and provide evidence that HCV replicates in macrophages, B cells, T cells, and other extrahepatic tissues. We also analyzed both short term and long term in vitro systems used to culture HCV. These systems vary in their purposes and methods, but long term culturing of HCV in B cells, T cells, and other cell types has been used to analyze replication. It is therefore now possible to study HIV-HCV co-infections and HCV replication in vitro.

Successful Treatment of Fibrosing Cholestatic Hepatitis After Liver Transplantation

Background A minority of liver transplant (OLT) recipients with hepatitis C virus (HCV) develop fibrosing cholestatic hepatitis (FCH), a severe form of HCV recurrence associated with early graft failure and death. There are few reports of successful salvage strategies. In this retrospective study, we sought to determine the characteristics and outcomes for patients with FCH at our transplant center. Methods All cases of HCV-positive OLT recipients from July 2007 through July 2010 were reviewed. Patient demographics, donor characteristics, and the post-OLT clinical course were analyzed. Tacrolimus-based immunosuppression was used. FCH was treated by conversion to cyclosporine A (CsA) and aggressive treatment with pegylated interferon (IFN) alpha2A and ribavirin (RBV). Liver biopsies and HCV RNA were obtained frequently per protocol or for cause. Results The rate of FCH during the study period was 13.5% (5/37). Of the 5 patients with FCH (4 males, 4 Caucasian), mean age was 51 (± 4.8) years and the Model for End-Stage Liver Disease (MELD) score at listing was 26.6 (± 10). Three of the 5 received liver and kidney (L/K) transplants (60%); the rate of L/K transplant in non-FCH patients was 12.5%. HCV RNA levels ranged from 5 to 6.69 log IU/mL pre-OLT; none were on anti-HCV therapy at the time of OLT. Mean ischemic time was 385 (± 152) minutes; donor age was 34.4 (± 13.7) years. No CMV infections developed postoperatively. Time to histologic HCV recurrence was 2 (± 2.23) months (range, 1–6); FCH occurred at 2.2 (± 2.2) months. Patients were converted from tacrolimus to CsA and treated with IFN and RBV; 2 were changed to consensus IFN. HCV RNA increased post-OLT in all, but responded to therapy in 4 of 5. None of the L/K recipients experienced renal graft rejection during treatment. Four of 5 had clinical and histologic improvement; 1 progressed to cirrhosis with minimal inflammation. One-year patient survival after OLT in this group was 80%. Liver allograft rejection occurred in 60% at 4.7 (± 5.5) months and was treated by CsA and prednisone dosage adjustments. In this cohort of patients undergoing OLT for HCV, FCH occurred early after OLT but responded to aggressive management with conversion from tacrolimus to CsA and treatment with pegylated IFN or consensus IFN/RBV. There was a higher rate of combined L/K transplants in the FCH group compared with the non-FCH group. Liver allograft rejection occurred in 60% of cases, but responded to treatment in all; no renal graft rejection occurred in the 3 with L/K transplants while on IFN. One-year graft and patient survival was 80%. Conclusion Better survival with FCH is possible with early initiation of IFN/RBV therapy with close monitoring of biopsies and viral load, and conversion from tacrolimus to CsA. Treatment can be performed even in L/K transplantation recipients, although it is associated with a higher incidence of treatable liver allograft rejection.

GB virus C infection among young, HIV‐negative injection drug users with and without hepatitis C virus infection

Our study examined the association between GB virus C (GBV-C) and (i) hepatitis C virus (HCV) infection status, (ii) biomedical indicators of liver disease (alanine and aspartate aminotransferases) and (iii) HCV RNA level among young injection drug users (IDUs) recruited using street outreach and respondent-driven methods. Cross-sectional and longitudinal analyses were completed. GBV-C (active or resolved) infection was significantly (P < 0.05) more prevalent among HCV antibody-positive (anti-HCV+) (65.1%) than antibody-negative (anti-HCV-) (32.3%) (OR = 3.9, 95% CI: 2.3-6.9) IDUs. The prevalence of resolved GBV-C infection was highest among those with chronic HCV infection (41.9%), followed by those with resolved HCV infection (34.4%) and significantly lower (P < 0.05) among anti-HCV participants (16.9%). Although not statistically significant (P = 0.13), a similar pattern was observed for active GBV-C infection. No association between GBV-C infection status and biomedical indicators of liver disease or HCV RNA level over time was observed. In conclusion, GBV-C infection prevalence was higher among anti-HCV+ compared to anti-HCV- young IDUs, similar to prior studies among older populations. In particular, chronically HCV-infected young IDUs had an increased rate of GBV-C clearance.

No evidence of occult hepatitis C virus (HCV) infection in serum of HCV antibody-positive HCV RNA-negative kidney-transplant patients

Persistence of hepatitis C virus (HCV) in patients who cleared HCV is still debated. Occult HCV infection is described as the presence of detectable HCV RNA in liver or peripheral blood mononuclear cells (PBMCs) of patients with undetectable plasma HCV-RNA by conventional PCR assays. We have assessed the persistence of HCV in 26 kidney-transplant patients, followed up for 10.5 years (range 2-16), after HCV elimination while on hemodialysis. If HCV really did persist, arising out of the loss of immune control caused by institution of the regimen of immunosuppressive drugs after kidney transplantation, HCV reactivation would have taken place. Their immunosuppression relied on calcineurin inhibitors (100%), and/or steroids (62%), and/or antimetabolites (94%). An induction therapy, given to 22 patients, relied on rabbit antithymocyte globulin (59%) or anti-IL2-receptor blockers (32%). All patients had undetectable HCV RNA as ascertained by several conventional tests. At the last follow-up, no residual HCV RNA was detected in the five liver biopsies, the 26 plasma, and in the 37 nonstimulated and 24 stimulated PBMCs tested with an ultrasensitive RT-PCR assay (detection limit, 2 IU/ml). No biochemical or virologic relapse was seen during follow-up. The absence of HCV relapse in formerly HCV-infected immunocompromised patients suggests the complete eradication of HCV after its elimination while on dialysis.

Real-time Quantitative Assay for Routine Testing of HCV RNA in Formalin-fixed, Paraffin-embedded Liver Samples

The assessment of hepatitis C virus (HCV) RNA in liver tissues is clinically relevant in cases where histology, liver function tests, and HCV serology are not sufficient for a definitive diagnosis of HCV-related hepatitis. We analyzed 215 formalin-fixed, paraffin-embedded liver needle biopsies from patients infected with HCV genotypes 1b and 2. HCV RNA extracted from paraffin sections were quantified by means of a TaqMan real-time reverse transcription-polymerase chain reaction method. The quantification of HCV RNA in liver tissue was correlated with the amount of HCV detected by immunohistochemistry (IHC) on paired frozen biopsies, the HCV RNA load in the serum, and the main serum tests of liver function and cholestasis. HCV RNA was detected by real-time reverse transcription-polymerase chain reaction in 169 liver biopsies (78.6%) with a mean value of 13.59+/-37.25 IU/ng. Tissue HCV RNA levels strongly correlated with the IHC results (P<0.001, Spearman test), HCV serum load (P<0.001), aspartate aminotransferase (P=0.001), gamma-glutamyl transpeptidase (P=0.012), and aspartate aminotransferase/alanine aminotransferase ratio (P=0.029). HCV RNA was amplified in up to 7-year-old archival tissue samples. Real-time HCV RNA quantification on archival liver tissue may be clinically relevant in case of "occult" HCV infection or for the diagnosis of patients with known HCV infection and hepatic dysfunction but seronegative for HCV RNA. The assessment of the levels of HCV RNA in the liver might also be important for monitoring the effectiveness of antiviral therapy and the progression of disease in patients with chronic HCV hepatitis.

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.

Hepatitis C virus replication in patients with occult hepatitis C virus infection†

We read with interest the review by Welker and Zeuzem1 on occult hepatitis C virus (HCV) infection. In this review, the authors claim that viral replication has not been proven in occult HCV infection. However, the presence of the antigenomic HCV RNA strand has been demonstrated in liver and in peripheral blood mononuclear cells (PBMCs) of patients with occult HCV who are negative for anti-HCV and serum HCV RNA2 (and references in Carreño et al.2). There is no doubt that detection of the antigenomic strand is an indicator of viral replication as recognized by Welker and Zeuzem in their review.1 Furthermore, ultracentrifugation of serum samples before performance of real-time reverse transcription polymerase chain reaction (RT-PCR) detects very low levels of viremia in patients with occult HCV infection, and the density of the circulating HCV particles was identical to those found in the serum of patients with chronic hepatitis C.2 In addition, family members of patients with occult HCV infection show serological markers of HCV infection even more frequently than those of patients with chronic hepatitis C.3 Other authors have reported that low-level persisting HCV remains infectious.4 Finally, HCV-specific CD4+ T cell proliferative responses to nonstructural 3 and/or 4 proteins have been detected among patients infected with occult HCV,2 which may not have occurred unless there was ongoing HCV replication, either within the liver or in immune-privileged sites. So, taking all these data into account, one may convincingly assume that HCV replication takes place in patients with occult HCV infection who no doubt are potentially infectious. However, Welker and Zeuzem1 review two studies that unconvincingly failed to detect occult HCV infection in cryptogenic hepatitis,5, 6 because of several concerns with the methodology employed. Thus, one study5 used whole blood to assess occult HCV infection, but it was demonstrated in patients with occult hepatitis C that the sensitivity of HCV RNA detection substantially decreased when using whole blood instead of PBMCs.2 Another study6 included a very small and heterogeneous population, and these authors did not perform detection of HCV RNA either in liver or in serum by PCR after ultracentrifugation. Thus, their results do not exclude the existence of occult HCV infection. On the other hand, an anti-HCV core antibody assay has been developed recently, which adds another diagnostic tool because it has been proposed as a potential new marker of the serologically silent form of occult HCV infection.7 In contrast, other studies have confirmed the existence of occult HCV infection by detection of HCV RNA in liver.8, 9 Furthermore, contrary to what is concluded by Welker and Zeuzem, HCV proteins have been detected in liver10 and in lymph nodes11 of patients who are negative for anti-HCV and serum HCV RNA. In summary, considering the data available, there is cumulative evidence strongly supporting the idea that HCV replication takes place in patients with occult HCV infection. Therefore, in order to diagnose occult HCV infection among these patients, it is mandatory to perform appropriate molecular and immunoserological tests in serum, PBMCs, and liver biopsies. Vicente Carreño*, Javier Bartolomé*, Inmaculada Castillo*, Juan Antonio Quiroga*, * Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.

Eradication of Hepatitis C Virus in Patients Successfully Treated for Chronic Hepatitis C

It is unclear whether hepatitis C virus (HCV) is eradicated in patients with chronic hepatitis C who achieved a sustained virologic response (SVR). In this long-term follow-up study, including chronic hepatitis C patients who achieved SVR after interferon-based therapy, the presence of residual HCV RNA in serum, liver, and peripheral blood mononuclear cells (PBMCs) was assessed, using transcription-mediated amplification (sensitivity, <9.6 IU/mL). The benefit of SVR on liver fibrosis was evaluated using the METAVIR score. A total of 344 patients were followed up for a median duration of 3.27 years (range, 0.50-18 y; interquartile range [IQR], 1.68-5.35 y). A total of 114 patients had a posttreatment liver tissue (median, 0.50 y; range, 0-14 y; IQR, 0-3.5 y) and a total of 156 had one PBMC (median, 3.0 y; range, 0.50-18 y; IQR, 1.25-5.50 y). 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. Histologic analysis of 126 paired pretreatment and posttreatment liver biopsy specimens (median, 0.50 y; range, 0-14 y; IQR, 0-3.5 y) showed that fibrosis stage was improved in 56%, stable in 32%, deteriorated in 12%. Regression of cirrhosis was observed in 9 of 14 (64%) (95% confidence interval, 39-89) patients. No cirrhosis decompensation was observed, and 3 patients developed hepatocellular carcinoma. In this large cohort of chronic hepatitis C patients, SVR was durable up to 18 years after treatment cessation, in addition to fibrosis stability/improvement (88%) and cirrhosis regression (64%). The presence of residual HCV RNA was observed only in liver tissue (1.7%). This result strongly suggests that SVR may be considered to show eradication of HCV infection.

Distinct hepatitis C virus core and F protein quasispecies in tumoral and nontumoral hepatocytes isolated via microdissection

Hepatitis C virus (HCV) genetic variability may be involved in liver carcinogenesis. We investigated HCV core and corresponding putative F protein genetic variability in hepatocellular carcinoma (HCC) and cirrhotic nodules. Hepatocyte clusters from 7 patients with HCC and HCV1b-related cirrhosis were isolated via microdissection of HCC tissues and 2 nontumoral cirrhotic nodules. The HCV core complementary DNA was cloned and sequenced from each liver compartment and from the serum of 2 patients. Nucleotide diversity and synonymous and nonsynonymous substitutions were analyzed within and between compartments via phylogenetic analysis and Mantel's test. Liver HCV RNA accumulation was lower in HCC. Increased quasispecies diversity and complexity was observed with HCC in 6 of 7 patients. Mantel's test demonstrated marked compartmentalization of quasispecies between HCC and cirrhotic nodules in all 7 patients and also between the 2 nontumoral nodules in 5 of them. Synonymous-nonsynonymous substitution analysis indicated low selection against tumoral core quasispecies in all patients and a more selective pressure against F protein quasispecies in all compartments. In the 2 subjects analyzed, HCC and nontumoral hepatocyte quasispecies were only minor or undetected in serum. In tumoral hepatocytes, low-replicating hepatitis C quasispecies are compartmentalized and more diversified and are subjected to low selective pressure. Our study supports the importance of core genetic variability in hepatocellular carcinogenesis.

Cellular Immune Responses Associated with Occult Hepatitis C Virus Infection of the Liver

Occult hepatitis C virus (HCV) infection is a type of recently identified chronic infection that is evidenced only by detection of HCV RNA in liver; patients consistently test negative for antibodies to HCV and HCV RNA in serum. Using ex vivo and in vitro measures of T-cell responses, we have identified functional virus-specific memory CD4(+) and CD8(+) T cells in the peripheral blood of patients with occult HCV infection. The features of the virus-specific T cells were consistent with immune surveillance functions, supporting previous exposure to HCV. In addition, the magnitudes of CD4(+) and CD8(+) T-cell responses were in parallel and correlated inversely with the extent of liver HCV infection. The detection of HCV-specific T cells in individuals in whom HCV RNA can persist in the liver despite the absence of viremia and antibodies indicates that HCV replication is prolonged in the face of virus-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that HCV-specific cellular immune responses are markers not only of previous exposure to and recovery from HCV but also of ongoing occult HCV infection.