Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R ² = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.     

Presence of viral replicative intermediates in the liver and serum of patients infected with hepatitis C virus

Hemophilic patients may present immunological dysfunctions resulting from either human immunodeficiency virus (HIV) infection, or other factors like impure factor VIII concentrate and other viral infections. We evaluated prospectively the serologic response to polio vaccination of Israeli hemophilic patients who were vaccinated during an outbreak of poliomyelitis. Eighty-two hemophilic patients, 43 seronegative and 39 seropositive for human immunodeficiency virus (HIV), were vaccinated with enhanced inactivated poliovirus (elPV). Titers of antibodies for poliovirus types 1–3 were determined before and 4 weeks after immunization. T helper and suppressor lymphocytes (T4 and T8), B and T lymphocyte mitogenic response, and natural killer cells were tested and correlated with the response to vaccination. Both groups responded to vaccination with increased titers of antibodies to the three viral types, 4 weeks after immunization. HIV-seronegative patients, however, exhibited higher titers than the HIV-seropositive group. The same pattern was found when 21 patients were tested 1 year after the exposure to elPV. HIV seropositive patients were grouped according to their T4 count (between 16/μ and 500/μ). There was no statistically significant difference in the response of these different groups to vaccination. No correlation was found between the response to vaccination and other immune parameters. These results suggest that asymptomatic HIV-seropositive hemophilic patients respond well to elPV, irrespective of their T4 count. © 1993 Wiley-Liss, Inc.     

Hepatitis C virus RNA in patients with chronic hepatitis C

Reviews recent data on the detection of genome and replicative HCV RNA in patients with chronic hepatitis C by PCR and in situ hybridization. Discusses the results of HCV RNA detection in the liver, lymphocytes, serum, and other organs and tissues and notes the relationship between the incidence of RNA and activity of the pathological process. Analyzes the results of HCV RNA detection after IFN treatment. Discusses the role of HCV RNA in the pathogenesis of hepatitis C.     

Factors affecting serum concentrations of hepatitis C virus (HCV) RNA in HCV genotype 1-infected patients with chronic hepatitis

The serum concentration of hepatitis C virus (HCV) RNA is usually stable (4 to 8 log(10) IU/ml) in untreated patients with chronic hepatitis C. While this baseline HCV RNA concentration ([HCV RNA](BL)) is predictive of a sustained virologic response to treatment, its determinants are only partially identified. We therefore analyzed the baseline characteristics of 2,472 HCV genotype 1-infected patients to identify correlations with gender, age, race, weight, body mass index (BMI), HCV acquisition mode, HCV subtype, alanine aminotransferase concentration, or histopathologic changes in the liver. After separation of the data according to four [HCV RNA](BL) groups (< or =5.0, >5.0 to 5.6, >5.6 to 5.9, and >5.9 log(10) IU/ml), we determined that increasing [HCV RNA](BL) correlated (P < 0.05) with increasing proportions of patients who were male, >40 years of age, or heavier (a weight of >85 kg or a BMI of >27 kg/m(2)). Histologic activity index (HAI) data were available for 1,304 of these patients: increasing [HCV RNA](BL) correlated with higher fibrosis and necrosis-inflammation scores. As a continuous variable, [HCV RNA](BL) correlated with age, gender, weight (continuous or < or =85 versus >85 kg), BMI (continuous or < or =27 versus >27 kg/m(2)), subtype, fibrosis score, and necrosis-inflammation score; however, multiple-regression analysis yielded P values of <0.1 only for age, gender, BMI (< or =27 versus >27 kg/m(2)), and fibrosis score. While our findings are suggestive of a role for these factors in maintenance of the pretreatment state of HCV infection, the multiple-regression model accounted for only < or =4.6% of the [HCV RNA](BL) differences between individuals (R(2) = 0.046 for 1,304 patients with HAI scores; 0.043 for all 2,472 patients).     

Real-time PCR assays for hepatitis C virus (HCV) RNA quantitation are adequate for clinical management of patients with chronic HCV infection

Because of the use of viral kinetics during polyethylene glycol (PEG)-interferon-ribavirin therapy and the development of specific new anti-hepatitis C virus (anti-HCV) drugs, assessment of the efficacy of anti-HCV drugs needs to be based not on end-point PCR assays but on real-time PCR. The aim of this study was to determine if the two available commercial real-time PCR assays, the Abbott RealTime HCV assay and the Roche Cobas TaqMan HCV assay, can become the standard for HCV RNA quantification. We investigated the prognostic relevance of HCV RNA viral loads at baseline, week 4, and week 12 to a rapid and early virological response to antiviral therapy by using the two assays. Of 59 na?ve patients chronically infected by HCV (41 infected with genotype 1) who were treated with ribavirin plus PEG-interferon alfa-2b for 48 weeks, 24 patients (41%) showed a sustained virological response (SVR). With the two assays, viral loads were highly correlated, irrespective of genotype (R2=0.94 for all cases). No difference in diagnostic value was found between the Abbott and Roche assays at week 4, with respective negative predictive values (NPVs) of 84% and 78% and positive predictive values (PPVs) of 62% and 56% (not significant), and at week 12, the respective NPVs were 91% and 90% and PPVs were 44% and 46% (not significant). At week 12, 83% (20/24) and 96% (23/24) of patients with SVR tested negative for HCV RNA by the Abbott and Roche assays, respectively (the difference is not significant). In conclusion, the high sensitivities and large dynamic ranges of the Abbott and Roche assays show that a single real-time quantitative PCR assay is fully adequate for clinical and therapeutic management of HCV.     

Long-term follow-up of hepatitis B virus and hepatitis C virus replicative levels in chronic hepatitis patients coinfected with both viruses

Dual infection with hepatitis B and C viruses is often encountered in endemic areas of both viruses. However, understanding of the clinical and virological implications is limited. The aim of this study was to investigate the role of each virus in liver injury and the interaction between the two viruses in dual infection with hepatitis B and C viruses. Three patients who had chronic infection with both hepatitis B and C viruses were examined, and a longitudinal study of both serum hepatitis B virus DNA and hepatitis C virus RNA levels over 4 years was undertaken. The results were correlated with serum alanine aminotransferase levels. Serum alanine aminotransferase values showed a relationship with hepatitis B virus replicative levels, but not with hepatitis C virus replicative levels in all 3 patients. Serial changes of replicative levels of both viruses were studied, and it was found that hepatitis C virus replicative levels were enhanced after the decline of hepatitis B virus replication in 1 of the 3 patients. In the remaining 2 patients, a transient rise of hepatitis C virus replicative levels in association with a decrease of hepatitis B virus replication was also observed during part of the follow-up period. These findings indicate that hepatitis B virus may play a dominant etiological role in liver injury, and that a suppressive action between hepatitis B and C viruses may occur in dual infection with both viruses. © 1995 Wiley-Liss, Inc.     

Immunochemical identification and detection of serum fibronectin in liver fibrosis patients with chronic hepatitis C

Serum tests measuring the dynamic processes of fibrogenesis and fibrolysis may reflect the severity of liver disease. Fibronectin plays a role in liver fibrosis. The aim of this study was to assess the diagnostic value of fibronectin in chronic HCV infection among Egyptian patients. Fibronectin was identified using specific monoclonal antibody and Western blot at 90-kDa in sera of HCV infected patients with liver fibrosis. The purified serum fibronectin showed one peak at 8 min when analyzed by capillary zone electrophoresis. Fibronectin was quantified in serum using ELISA. The mean (+/-SD) serum level of fibronectin (mg/L) in liver fibrosis patients were 450.9 (+/-170.3) and 230.5 (+/-90.3) in control individuals, respectively. There was a significant correlation between METAVIR score and serum fibronectin (r=0.401; P<0.0001). The area under the receiver operating characteristic (ROC) curve of fibronectin for discriminating patients with liver fibrosis from those with no fibrosis livers and its p value were 0.78 and P<0.0001. The efficiency of fibronectin for discriminating patients with liver fibrosis from those with non fibrosis livers was 75%. In conclusion, serum fibronectin can differentiate HCV infected patients with liver fibrosis from patients with non fibrosis.     

A reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus RNA

We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.     

Simultaneous amplification and detection of specific hepatitis B virus and hepatitis C virus genomic sequences in serum samples

A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169–7126).     

Quantitative detection of hepatitis C virus RNA by light cycler PCR and comparison with two different PCR assays

The new Light Cycler technology was adapted to the detection of hepatitis C virus (HCV) RNA in clinical samples. Sera from 81 patients were tested by Light Cycler PCR, AMPLICOR HCV Monitor assay, and in-house PCR. Our data demonstrate that Light Cycler is a fast and reliable method for the detection and quantitation of HCV RNA.     

Detection of hepatitis C virus RNA in serum and peripheral blood mononuclear cells in patients with chronic hepatitis C treated with interferon alpha

PCR was used to detect hepatitis C virus (HCV) RNA in serum and peripheral blood mononuclear cells (PBMCs) for evaluation of a six-month course of Interferon therapy in 18 patients with histologically confirmed chronic hepatitis C. At follow-up six months after the end of therapy positive-stranded (genomic) and negative-stranded (anti-genomic, presumptive replicative intermediate) HCV RNA could be detected in PBMCs of all ten patients who either did not respond to therapy or suffered a relapse; genomic strand RNA was detected in five patients who responded but then relapsed. The study confirms that interferon therapy leads to inhibition of HCV replication but not eradication of the virus. Persistence of the virus at extrahepatic sites may explain its reactivation after cessation of interferon therapy.     

GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum.

PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.     

Correlation between detection of antibodies against hepatitis C virus in oral fluid and hepatitis C virus RNA in serum

Reported here are the results of a study designed to determine the correlation between hepatitis C virus (HCV) RNA positivity in serum and the detection of antibodies against HCV in oral fluid by testing paired serum/oral fluid samples. For the 85 serum samples found positive for antibodies against HCV, using a screening assay and a confirmation assay, 70 of the corresponding oral fluid samples tested positive for HCV antibodies using a previously modified screening assay. For 52 of the 59 serum samples found positive for HCV RNA, the corresponding oral fluid samples also tested seropositive for HCV, while 18 of the 26 serum samples that were negative for HCV RNA had corresponding oral fluid samples that tested seropositive for HCV. For the control group of 54 serum samples that were negative for HCV antibodies, all of the corresponding oral fluid samples were also negative for HCV antibodies, while 53 of the serum samples tested negative for HCV RNA. These results suggest that HCV antibody detection in oral fluid has a slightly higher sensitivity when used to test patients whose serum samples are positive for HCV RNA (chi-square test, p=0.035; Mid-P exact, p=0.049).     

Immunohistochemical detection of hepatitis C virus antigen in paraffin embedded liver biopsies from patients with chronic liver disease

A simple and reproducible immunohistochemical (IHC) staining method that adequately identifies and localizes hepatitis C virus (HCV) in human livers is still not available. We performed IHC staining using both a new monoclonal antibody against HCV and a polyclonal human anti-HCV IgG to 94 liver biopsies from HCV seropositive patients with chronic hepatitis and 15 control liver biopsies. Positive nuclear staining was consistently observed predominantly in hepatocytes and much less in lymphocytes with either antibody in 57% of anti-HCV seropositive cases but in none of the controls. However, the monoclonal antibody yielded a stronger positive reaction and in a greater proportion of hepatocytes. In 78% of the positive cases, more than a quarter of the hepatocytes showed nuclear staining. The degree of hepatic HCV load as revealed by intensity and extent of positive staining did not correlate with histological changes in the liver. The new monoclonal antibody against HCV appeared to be suitable for identifying HCV in tissues by a simple IHC stain and can be used to explore the pathogenesis of liver injury induced by this virus.