Hepatitis C Virus RNA Levels Determined by Branched DNA Probe Assay Correlated with Levels Assessed Using Competitive PCR

Objective: To search for comparative differences, levels of hepatitis C virus (HCV) RNA were examined by branched DNA (bDNA) probe assay and by competitive polymerase chain reaction (PCR). Methods: The study population included 234 patients (chronic hepatitis 146, cirrhosis 36, hepatocellular carcinoma 52), all of whom were positive for HCV RNA, as determined by PCR. We quantified HCV RNA levels of all serum samples by both bDNA probe and competitive PCR. Results: HCV RNA was detected in serum samples by bDNA assay in 142 (60.7%) of the 234 patients; this rate was significantly higher in 106 (73.6%) of the 144 patients of genotype II than in 20 (41.7%) of 48 of genotype III and in 16 (38.1%) of 42 of genotype IV (p < 0.001, respectively). The median HCV RNA levels by bDNA assay (×10⁶eq/ml) were 0.1,0.1,0.4,1.4, and 5.3 among patients with HCV RNA levels <3, 4, 5, 6, and 7, respectively, by competitive PCR (logarithmic transformation copy numbers/50 μl). A significant correlation was found between HCV RNA levels by bDNA and competitive PCR ( r = 0.5747, p < 0.001). There was a correlation among patients of genotype II and genotype III but not genotype IV. Conclusion: We recommend bDNA assay for use in clinical practice because the procedure is not difficult and is less contamination-prone. The HCV RNA levels determined using this assay correlated with those examined by competitive PCR.     

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA

Summary. There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme-labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 10⁷–10⁸ molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%. Lastly, bDNA showed the loss of HCV RNA in six out of six evaluable patients who had complete biochemical responses to interferon; five out of six non-responders also showed appreciable declines in HCV RNA level, but in only two did HCV RNA drop below the detection limit; these two cases remained PCR positive. Seventeen placebo-treated patients did not lose HCV RNA by either bDNA or PCR. Hence the bDNA assay is a practical means to measure HCV RNA in a variety of clinical settings. Although it is not as sensitive as PCR, it has greater specificity, is directly quantitative, and can be used in any routine laboratory that can perform microwell EIAs. This simplified quantitation may be of particular benefit in evaluating the probability of HCV transmission and the response to anti-viral therapy.     

Viral and host factors in the prediction of response to interferon-α therapy in chronic hepatitis C after long-term follow-up

Acute infection with hepatitis C virus (HCV) develops into a chronic hepatitis in about 50–70% of patients. Treatment of these patients with interferon-α (IFN-α) results in a sustained long-term response in only 15–20% but causes numerous unwanted side-effects in a higher percentage of patients. The aim of our study was to define host or viral parameters that would allow identification of responders and non-responders to IFN-α prior to the onset of treatment. We studied a group of 87 patients suffering from chronic hepatitis C who were treated with IFN-α. After long-term follow-up, 18 patients (21%) showed a sustained response to IFN-α therapy (normalization of serum transaminases and loss of viral RNA from serum) for up to 7 years after therapy had ceased. By univariate and multivariate analyses, no host factors were found to be predictive of response to therapy. Neither the degree of inflammation or fibrosis in liver biopsy samples obtained before treatment nor immunogenetic factors (major histocompatibility complex II haplotype and tumour necrosis factor-α promoter polymorphism) were associated with response to therapy. In contrast, viral parameters showed a strong association with response to therapy. HCV genotype 3 was found significantly more frequently in responders (P = 0.034), and mean HCV RNA concentration was lower in responders (3.1 × 10⁴) than in non-responders (2.5 × 10⁵) (P = 0.01). By multivariate analysis, both HCV genotype and HCV RNA concentration were independent predictors of response to therapy. However, exact prediction of response to treatment for an individual patient was not possible on the basis of pretreatment viral RNA concentration or viral genotype. The best association with response to therapy was found to be clearance of HCV RNA from serum 3 months after the start of treatment (32 of 34 partial and sustained responders vs 0 of 53 non-responders; P = 0.001). In conclusion, determination of pretreatment viral factors, but not host factors, was significantly correlated with treatment response but did not give an accurate prediction for patients, whereas clearance of HCV RNA from serum after 3 months of therapy was predictive of response to therapy.     

Interferon-γ brings additive anti-viral environment when combined with interferon-α in patients with chronic hepatitis C

T-cell hyporesponsiveness may lead to chronicity of hepatitis C virus (HCV) infection. We evaluated whether interferon (IFN)-γ injection can bring a Th1-dominant environment to patients with chronic hepatitis C. Seventeen patients with genotype 1b received natural IFN-α 5MU daily for the first 2 weeks and three times a week for the next 22 weeks followed by natural IFN-γ 1 MU daily for 2 weeks. In 4 of 17 patients (23.5%), alanine aminotransferase (ALT) was normalized and 3 of these 4 patients (75.0%) cleared HCV RNA. β2 microglobulin (BMG), neopterin and soluble (s) Fas increased with IFN-α and increased more with IFN-γ. Serum interleukin (IL)-12, CD4 and CD8 remained unchanged with IFN-α but increased after IFN-α was replaced by IFN-γ. IL-10 was not changed either with IFN-α or γ. Productions of IL-2, IFN-γ and tumor necrosis factor (TNF)-α by peripheral blood mononuclear cells did not change by IFN-α therapy, however, they were enhanced at the end of IFN-γ therapy. Productions of IL-2 and 4 were unaffected. These results show that some immune parameters become Th1-dominant by additional IFN-γ in patients with chronic hepatitis C. Combination of these two IFNs should be explored.     

丙型肝炎病毒全长基因嵌合萤光素酶重组质粒转染细胞系的建立与初步应用

目的 构建能产生较高效价重组病毒的HCV细胞感染模型,为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养系统.方法 利用PCR技术在HCV NS5A C端插入海肾萤光素酶( Renilla Luciferase,Rluc)报告基因,并引入能提高HCV效价的突变,酶切鉴定重组基因序列构建成功后,转染入...     

Low HCV replication levels in end-stage hepatitis C virus-related liver disease.

BACKGROUND/AIMS: The relationship between HCV RNA levels and the severity of HCV-related liver disease has been addressed in a few studies, which has led to conflicting results. To clarify this point, we studied serum HCV RNA levels in patients with HCV liver disease at various stages, using a second-generation branched DNA (bDNA) assay. METHODS: One hundred and forty-eight patients with chronic HCV infection were classified into 3 groups: group A included 92 patients with chronic active hepatitis (CAH) without cirrhosis; group B included 30 patients with CAH and compensated cirrhosis; group C included 26 patients with end-stage cirrhosis. In all patients, serum HCV RNA was sought by qualitative PCR and quantified using second-generation bDNA assay. HCV RNA was also quantified after liver transplantation in 22 patients from group C. HCV genotype was determined in all patients. RESULTS: HCV RNA was detected by PCR in 100%, l00% and 92% of the patients from groups A, B and C, respectively (NS). The proportion of patients with HCV RNA levels higher than the cut-off of bDNA assay was significantly lower in patients from group C than in patients from groups A and B (50% vs 94% and 93% respectively, p<0.0001). The mean HCV viremia was lower in group C than in groups A and B (1.35+/-0.24 MEq/ml vs 5.00+/-6.04 MEq/ml and 5.85+/-7.70 MEq/ml, respectively, p<0.0001). This difference was independent of HCV genotype. In the patients from group C, post-transplant HCV RNA levels were significantly higher than pretransplant HCV RNA levels (14.90+/-26.40 vs 1.35+/-0.24 MEq/ml, p=0.0065). CONCLUSIONS: HCV RNA levels do not appear to differ significantly among patients with CAH with or without compensated cirrhosis. In contrast, HCV RNA levels seem to be significantly lower in patients with end-stage HCV-related liver cirrhosis. In these patients, high levels of replication are restored after liver transplantation, suggesting that low pretransplant viral loads are not due to the intrinsic characteristics of the infective viral strains, but rather to the severity of liver disease.     

Comparison between three quantitative assays in patients with chronic hepatitis C and their relevance in the prediction of response to therapy

To compare three quantitative assays measuring viral load in patients with chronic hepatitis C and to determine their value in predicting response to interferon (IFN) therapy, we analysed serum from 896 patients from eight European Centres using QUANTIPLEX^™ bDNA, MONITOR AMPLICOR^™ and SUPERQUANT^™ assays. Analyses were performed on the same sample. Viral genotype was assessed using INNO-LiPA HCV II kits. Intercentre variations were observed that were related to the handling of specimens not processed and stored within 6 h of blood sampling. Among sera with optimal handling, a stronger correlation was observed between bDNA and SUPERQUANT (0.806) than between bDNA and MONITOR (0.677) and between MONITOR and SUPERQUANT (0.632). These discrepancies were greatest with genotype 2 (bDNA/SUPERQUANT= 0.772; bDNA/MONITOR=0.456; SUPERQUANT/MONITOR= 0.299). This correlation was influenced by viraemia level and was better at lower viral loads. The proportion of sera with undetectable viral load was 15% with bDNA, 9.7% with MONITOR and 7.7% with SUPERQUANT. For the three measurements, the best cut-offs of sustained response to IFN treatment were located at their detection threshold. Among patients with viral load below the detection level, a sustained response was observed in 35% tested with bDNA, 38% with MONITOR and 80% with SUPERQUANT. Hence a stronger correlation was observed between bDNA and SUPERQUANT than between either of these assays and MONITOR. SUPERQUANT was the most sensitive assay and this greater sensitivity was associated with a better predictive value of treatment response.     

Viral kinetics can predict early response to alpha-interferon in chronic hepatitis C

ABSTRACT— Aims/Background: Response rates to alpha-interferon therapy for chronic hepatitis C are poor. An early indication of efficacy would reduce the need for prolonged therapy, leading to significant cost savings. It was established that a change in quantitative hepatitis C virus RNA (HCV-RNA) titre at 4 weeks could predict the outcome of alpha-interferon therapy in chronic hepatitis C. Methods: Serum HCV-RNA titres were quantified using branched chain DNA (bDNA) assay in 26 patients who responded to alpha-interferon (serum HCV-RNA negative after 12 weeks therapy) and 11 age and sex matched non-responders. Quantitative bDNA and qualitative RT-PCR assays for HCV-RNA were measured pre-treatment and at 4 weeks. The change in quantitative HCV-RNA titre between pre-therapy and after 4 weeks was compared in the two groups. Results: Seventeen of the 37 patients had become RT-PCR negative at 4 weeks (early responders) and had an undetectable HCV-RNA titre on bDNA assay. Nine patients were RT-PCR positive at 4 weeks but negative by 12 weeks (delayed responders), and of these, 8 had an undetectable viral titre at 4 weeks on bDNA assay. The patient with a detectable HCV titre did become RT-PCR negative after 12 weeks, but subsequently became RT-PCR positive again at 24 weeks. All the non-responders had a detectable bDNA titre (>0.2 Meq/ml) at 4 weeks. Conclusions: Change in quantitative HCV-RNA titre measured by bDNA assay at 4 weeks predicts response to alpha interferon. If HCV-RNA remains detectable on bDNA assay at 4 weeks, no sustained response to treatment is found and alpha-interferon can be discontinued.     

Stability of hepatitis C virus RNA in serum from samples collected in a closed-tube system for serum separation and transport, as measured by a quantitative competitive PCR assay

SUMMARY. Recovery of hepatitis C virus (HCV) RNA, after variable time intervals from collection, was assessed using a closed-tube system for collection, separation and transport (SST^(tm) tubes). Blood from four hepatitis C-infected patients was collected in 12 SST^(tm) tubes and centrifuged within 1 to 3 h of collection. Tubes were then left 0, 8, 12, 24. 48 and 72 h at room temperature and at 4°C before removing serum. Hepatitis C virus RNA levels were measured by quantitative polymerase chain reaction (PCR) using the AMPLICOR HCV MONITOR^(tm) assay. Hepatitis C virus RNA levels in these samples were stable for at least three days at both temperatures. Polymerase chain reaction signals never decreased by more than 0.5 log. The reproducibility of the assays showed that the quantitative PCR method can be used with the storage conditions tested here. Our data suggests that processing blood in SST^(tm) tubes may be very useful in following hepatitis C virus RNA titres in infected patients, especially those receiving treatment.     

Antiviral effect of ribavirin in early non-responders to interferon monotherapy assessed by kinetics of hepatitis C virus RNA and hepatitis C virus core antigen

BACKGROUND/AIMS: To evaluate the efficacy of ribavirin, given in second intention in non-responders to interferon alone, by studying viral kinetics. METHODS: We conducted a trial including 203 patients with chronic hepatitis C, na?ve of treatment. Patients were treated with interferon three times a week with or without ribavirin and amantadine according to response. Viral kinetics were assessed by serial measurements of HCV RNA (bDNA 3.0 and Monitor 2.0) and a new assay, trak-C, able to quantify total Hepatitis C virus (HCV) core antigen. RESULTS: A significant initial drop in HCV RNA or HCV core antigen, under interferon alone, was associated with response to therapy, -4.85+/-1.33 log for HCV RNA in sustained responders versus -1.86+/-1.53 log for others groups, P<0.001. In patients receiving ribavirin in second intention, we also observed a similar drop in HCV RNA and HCV core antigen, predictive of sustained response, -2.67+/-1.26 log for HCV RNA in sustained responders versus -0.44+/-0.49 log in non-responders, P<0.001. CONCLUSIONS: Ribavirin has probably an additional antiviral effect in interferon treated patients. Kinetics of HCV RNA and HCV core antigen under treatment are highly predictive of a sustained virological response.     

Quantification of serum hepatitis C virus core protein level in patients chronically infected with different hepatitis C virus genotypes.

BACKGROUND/AIM: A novel fluorescent enzyme immunoassay (FEIA) for the detection and quantification of serum hepatitis C virus (HCV) core protein was developed. The aim of this study was to evaluate the relation among serum HCV core protein level, HCV RNA level, and HCV genotype in patients with chronic HCV infection. PATIENTS AND METHODS: Serum HCV core protein, HCV RNA, HCV genotype were determined in 175 patients using the FEIA, branched DNA assay (Quantiplex HCV RNA ver 1.0), and serologically defined genotyping assay, respectively. For the specificity, all 13 patients seronegative for anti-HCV were negative for serum core antigen and HCV RNA by FEIA and bDNA, respectively. RESULTS: FEIA assay seems to be more sensitive than bDNA for patients with HCV type 2 infection (detection: 83.4% v 63.4%, p < 0.01). There was a good overall correlation between the FEIA and bDNA results. However, when the patients were stratified into their HCV types, a correlation was observed in HCV type 1 but not in type 2 infection. Patients with HCV type 2 infection had a lower serum HCV core protein level (median 56 RFI) compared with type 1 infection (median 149 RFI, p < 0.01). Thirty seven patients subsequently received interferon alpha therapy, patients who showed a complete and sustained response had a lower pretreatment serum HCV core protein level compared with patients who had a relapse and nonresponders (36 v 338 RFI, p < 0.01). CONCLUSIONS: This study showed that FEIA (1) is a good assay for the detection and quantification of serum HCV core protein level, (2) is also very sensitive in detecting HCV core protein in patients with HCV type 2 infection, and (3) may have a role as a predictor of subsequent response to interferon therapy.     

Effect of interferon therapy on serum hepatitis C virus RNA levels in children with chronic hepatitis C

The majority of chronic hepatitis C in children was related to blood transfusion. We evaluated the efficacy and safety of interferon (IFN) treatment on 12 children with chronic hepatitis C who had serious underlying diseases. Natural IFN-α was administered in a dosage of 0.1 MIU/kg daily for 2 weeks and then three times a week for an additional 22 weeks. Serum HCV RNA levels, determined by multicyclic RT-PCR, were elevated in all subjects before treatments: the HCV RNA level was above 10⁷ copies/ml in 11 (91.7%) of 12 cases. After 24 weeks of therapy, HCV RNA was undetectable in all subjects. IFN was well tolerated by all children. Our results indicate that this IFN therapy is both beneficial and safe in children with chronic hepatitis C.     

Viral kinetics of hepatitis C virus RNA in patients with chronic hepatitis C treated with 18 MU of interferon alpha daily

BACKGROUND: A rapid decrease of hepatitis C virus (HCV) RNA is interferon (IFN) dose-dependent, and a 3-log decline of HCV-RNA is a strong predictor of sustained virological response. In this study, viral kinetics of HCV RNA in patients treated with 18 MU interferon alpha (IFN-alpha) daily for 2 weeks are presented. METHODS: Thirteen treatment-naive patients with chronic hepatitis C received 6 MU of IFN-alpha2a every 8 h for 2 weeks. Samples were obtained daily during the treatment period. HCV-RNA levels were determined using the quantitative VERSANT 3.0 bDNA assay (detection limit 520 IU/ml). When results were below the detection limit, HCV-RNA was measured by qualitative polymerase chain reaction (PCR) using the COBAS AMPLICOR HCV test, version 2.0 (detection limit of 50 IU/ml). RESULTS: In patients infected with genotype non-1, a 3-log decline of viral load was found 2.4 days after the start of induction therapy. Only one of three patients infected with genotype 1 had a 3-log decline in viral load within 14 days of the start of therapy. In four patients, a third phase of viral decline was observed. At the end of treatment, 10/13 (77%) and 7/13 (54%) patients were HCV-RNA-negative in quantitative assay and qualitative PCR, respectively. Only one of 13 patients achieved a sustained virological response (SVR). CONCLUSION: Daily administration of 18 MU IFN-alpha to patients infected with genotype non-1 induces a 3-log decline of viral load within 2.4 days of the start of treatment. In patients infected with genotype 1, only one-third of patients have a 3-log decline at 11 days.     

Viral clearance occurs very early during the natural resolution of hepatitis C virus infection in persons with haemophilia

We studied spontaneous hepatitis C virus (HCV) RNA clearance in 12 haemophilic patients. In their earliest anti-HCV positive samples, HCV RNA was undetectable in eight patients (66%), positive by polymerase chain reaction (PCR) but negative by branched-DNA (bDNA) in three others, and quantifiable by bDNA (4839 IU/mL) in only one patient. In contrast, in earliest anti-HCV positive samples from eight matched controls who had persistent viremia, HCV RNA was quantifiable by bDNA in seven (P = 0.0008) and at higher levels (range 4644-678 515 IU/mL; median 43 532 IU/mL). From initial HCV infection, HCV RNA cleared in 7 months or less in four patients and in 1-2 years in six others. HCV persisted for 5 years before clearance in the absence of repeated exposure in one patient. We conclude that HCV clearance usually but not always occurs within 1-2 years after infection and is more likely in those with lower than in those with higher early viral loads.