Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications

OBJECTIVE: To compare two recently developed molecular techniques for quantitating the levels of hepatitis C virus (HCV) RNA in the serum of patients with a wide spectrum of chronic hepatitis C. DESIGN: Serum samples from 299 patients with HCV viremia, 101 control patients without HCV infection, and 19 consecutive patients receiving systemic interferon therapy were evaluated by a commercially available branched-chain DNA (bDNA) assay and a quantitative competitive polymerase chain reaction (PCR). SETTING: University-based hepatology clinics and reference virology laboratory. PATIENTS: Patients with HCV viremia as defined by results of qualitative RNA PCR, including 53 HCV-infected blood donors, 34 patients receiving renal dialysis, and 212 patients attending a hepatology clinic. RESULTS: Results of in vitro and in vivo experiments indicated that the sensitivity and dynamic range of the PCR assays were greater than those of the bDNA assay. Detection of HCV viremia by the bDNA assay was highly dependent on viral RNA titers, with a sensitivity of 5% at HCV RNA titers of 5.0 logs per mL or less and 94% at titers of 5.5 logs per mL or greater. The best correlation between assays was observed in specimens with HCV RNA titers between 6.0 and 7.5 logs per mL (r = 0.73). In patients with high-titer HCV viremia, including liver transplant recipients and patients with cirrhosis, quantitative PCR results were an average of 12-fold higher than bDNA assay results. Results of repetitive testing of discordant specimens showed that these discrepancies were caused by a high kit-to-kit coefficient of variation (112%) in the bDNA assay. Of 19 patients receiving interferon therapy, 9 (47%) became bDNA negative, but only 5 became quantitative PCR negative. The bDNA-negative, quantitative PCR-positive patients all had relapse when therapy was discontinued. CONCLUSIONS: The bDNA assay has a narrower linear range for quantitation of HCV viremia than quantitative PCR. Because persons with low HCV titers may respond well to therapy, seropositive persons with negative bDNA results should be retested with PCR-based assays. Similarly, the bDNA assay may underestimate the true degree of HCV viremia in persons with end-stage infection (> 10(7) RNA equivalents/mL of sera). Despite these limitations, the combination of bDNA- and PCR-based assays appears to be optimal for selecting and following patients during interferon therapy.     

Post-hemorrhagic shock mesenteric lymph is cytotoxic to endothelial cells and activates neutrophils

The aim of this study was to evaluate the Chiron branched DNA (bDNA) assay for detection of serum hepatitis B virus (HBV) DNA in patients with chronic hepatitis B lacking hepatitis B e antigen (HBeAg) and undergoing interferon (IFN) therapy. Results obtained with the bDNA assay were compared with those obtained using the Abbott liquid hybridization (LH) assay and the polymerase chain reaction (PCR). Serial samples (274) from 34 patients were analysed. Analysis of variance results indicated that bDNA values were more significantly correlated than LH values with both PCR positive/negative results (probability of artifact (Prob > F) = 0.7 and 0.09 for LH and bDNA assays, respectively) and presence/absence of precore mutations (Prob > F = 0.21 and 0.001 for LH and bDNA assays, respectively). Both bDNA and LH results correlated highly with alanine aminotransferase (ALT) values (both had Prob > F values of 0.0) while PCR was not correlated with ALT (Prob > F = 0.05). In 26 evaluable patients, a model based on a generalized Knodell score was used to predict response to IFN therapy, as defined by normalization of ALT values during therapy. This model discriminated well between non-responders and responders. The bDNA results correlated well with the generalized Knodell score, while the LH results did not (Prob > F = 0.04 and 0.19 for the bDNA and LH assays, respectively). In conclusion, the bDNA assay appears to be useful for quantification of HBV DNA levels in HBeAg-negative chronic hepatitis as it correlates with biochemical and histological indications of disease severity as well as with response to IFN therapy.     

HCV RNA levels in serum, liver, and peripheral blood mononuclear cells of chronic hepatitis C patients and their relationship to liver injury

OBJECTIVE: We sought to evaluate the relationship between HCV RNA levels in serum, liver, and peripheral blood mononuclear cells (PBMC) and the degree of liver injury in chronic hepatitis C (CHC) patients. METHODS: Thirty-six consecutive CHC patients were included in the study. The liver damage was evaluated by the histological activity index (HAI) score. The HCV RNA levels in the three compartments studied were assessed by bDNA assay. Nineteen patients were treated with alpha-interferon 2b (IFN). RESULTS: Serum and liver HCV RNA levels in CHC patients were significantly associated with an increasing HAI score irrespective of the HCV genotypes. Cirrhotic patients showed higher HCV RNA levels than the CHC patients with HAI score 1-4 (p < 0.05), but had lower levels than the group with HAI score > 8 (p < 0.03). Patients with HAI score 1-4 showed the lowest levels of HCV RNA in PBMC. There was a strong relation (r = 0.78; p < 0.001) between serum and liver HCV RNA levels, but not between either serum or liver HCV RNA levels and those of PBMC. Seven patients showed a response to IFN and three of these had a sustained response. Pretreatment levels of HCV RNA in PBMC of the IFN responder patients were lower than those of the nonresponder patients (p < 0.02). CONCLUSIONS: The data indicate a relation between serum or liver HCV RNA levels and the degree of liver injury in CHC patients, and show that serum HCV RNA level mirrors the hepatic viral burden.     

Combination therapy with thymosin alpha1 and interferon for the treatment of chronic hepatitis C infection: a randomized, placebo-controlled double-blind trial

Hepatitis C is a major cause of liver disease leading to cirrhosis. Although interferon (IFN) is the only approved therapy, treatment is characterized by low response rates and dose-limiting side effects. We evaluated the addition of thymosin alpha1 (TA1), an immunomodulatory peptide, to the standard treatment regimen for hepatitis C to determine if combination therapy shows biological activity using outcome measures including normalization of alanine aminotransferase levels, histological activity, and viral load during treatment. We performed a randomized, double-blind, placebo-controlled trial to compare the biological activity of a combination TA1 and IFN with that seen for IFN alone in patients with chronic hepatitis C infection. One hundred nine patients were randomized for intention to treat and received 1.6 mg of TA1 subcutaneously twice weekly and 3 MU of IFN three times weekly; 3 MU of IFN three times weekly and placebo TA1; or placebo for both agents. All patients had chronic HCV infection with confirmation of chronic hepatitis on liver biopsy. Biochemical responders were followed up until alanine aminotransferase (ALT) levels became abnormal or for 26 weeks, and relapsers were retreated for 26 weeks in the same treatment arm. One hundred three patients completed treatment for 26 weeks, and six patients dropped out. The groups were similar with regard to sex, gender distribution, baseline histological activity index (HAI) score, risk factors, and viral titers. End-of-treatment biochemical response was seen in 37.1% of patients treated with combination therapy, 16.2% of patients treated with IFN alone, and 2.7% of untreated controls by intent-to-treat analysis (IFN/TA1 vs. IFN, chi2 = 4.05, P = .04). HCV RNA clearance was seen in 37.1% of IFN/TA1-treated patients and 18.9% of IFN-treated subjects. Mean HCV RNA titers were significantly lower than baseline at weeks 8, 16, and 24 after drug initiation among patients treated with IFN/TA1 but not in the other treatment arms. Histological improvement, as evidenced by a decrease in HAI of more than two points, occurred in the combination therapy arm more frequently than in comparison groups. Cumulative sustained biochemical responses were 14.2% and 8.1% in the IFN/TA1 and IFN arms, respectively, based on an intention-to-treat model. The combination of TA1 and standard IFN treatment for chronic hepatitis C showed evidence of biological activity at the completion of treatment by biochemical, histological, and virological outcome measures. Further research involving longer duration and varied dosing is needed.     

A PET study on cortical and subcortical control of pelvic floor musculature in women

The possibility of hepatitis B virus (HBV) infection in HBsAg-negative patients has been shown. However, an "inapparent" coinfection by HBV in hepatitis C virus (HCV)-positive patients generally is not taken into account in clinical practice. Mechanisms responsible for resistance to interferon (IFN) have not been completely clarified. The aim of this study was to investigate whether an "inapparent" coinfection by HBV in anti-HCV-positive chronic liver disease patients may influence IFN response. Fourteen anti-HCV positive, HBsAg-negative but serum HBV DNA-positive patients by PCR and 111 anti-HCV-positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis were treated with 3 MU of recombinant alpha-2a IFN 3 times weekly for 12 months. Serum HBV DNA and HCV RNA were determined before treatment, after 6-12 months and in coincidence with ALT flare-up by PCR. HBV PCR was performed using primers specific for the S region of the HBV genome and HCV PCR with primers localised in the 5'NC region of HCV genome. IgM anti-HBc was tested using IMx Core-M Abbott assay. By the end of treatment, ALT values had become normal in 4/14 HBV DNA-positive patients (28%), but all "responders" (4/4) relapsed between 2 and 5 months after therapy. All but one patient were HCV RNA-positive before treatment, 6 were also both HBV DNA and HCV RNA-positive during ALT flare-ups. In 5 patients, only HBV DNA and in 3 patients, only HCV RNA was detected when transaminase values increased. All patients remained HBsAg-negative and anti-HCV-positive. IgM anti-HBc was detected both before treatment and during ALT elevation in 3 patients and only during ALT relapse in 3 others. Of the 111 anti-HCV positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis, a biochemical response to IFN treatment was observed in 54% of the cases. Relapse of ALT values was observed in 47% of the cases during a follow-up of 1 year after treatment. "Inapparent" HBV/HCV coinfection may be implicated in cases of resistance to IFN treatment. In addition, HBV replication may persist in patients in whom HCV replication was inhibited by IFN treatment. The pathogenic role of HBV in liver disease was confirmed by detection of IgM anti-HBc in some cases; the appearance of these antibodies only after IFN treatment suggests that IFN may exert a selective role in favour of HBV. Further studies will show the effect of different treatment schedules. HBV DNA and/or IgM anti-HBc detection with very sensitive methods may be important both as a prognostic factor and as a tool for better understanding interviral relationships and mechanisms involved in multiple hepatitis virus infections.     

Factors influencing the response to interferon therapy in chronic hepatitis C. Studies on viral genotype and induction of 2',5'-oligoadenylate synthetase in the liver and peripheral blood cells

BACKGROUND: The mechanism behind the antiviral action of interferon (IFN) therapy in chronic hepatitis C virus (HCV) infection is not well understood, and, furthermore, few factors have been shown to be good predictors of a favourable response to IFN treatment in chronic HCV infection. METHODS: Freshly explanted liver cells and peripheral blood mononuclear cells (PBMC) from 80 patients with chronic HCV infection were used to study the capacity of IFN to induce the enzyme 2',5'-oligoadenylate synthetase (2'5'-AS) in vitro. The HCV genotype was determined in 53 patients. The induction of 2'5'-AS was correlated to the results of IFN-alpha treatment in 36 patients. RESULTS: Normalization of transaminases during IFN treatment was significantly associated with 2'5'-AS levels in liver cells cultured in the absence of IFN. A similar tendency, although not statistically significant, was found for IFN-induced levels of 2'5'-AS in liver cells. No such associations were found when PBMC were analysed. Six patients showed a sustained biochemical response. These six did not deviate significantly from the remaining patients with regard to base-line or IFN-induced levels of 2'5'-AS in liver cells or PBMC. Eradication of HCV RNA during IFN treatment did not correlate with 2'5'-AS levels in liver cells. Comparison of HCV genotype and clinical response showed that patients with genotype 3a had the most favourable outcome. No association was found between liver histology and treatment outcome. CONCLUSION: These data imply that direct effects of IFN on liver cells are of importance for the response to IFN treatment.     

Effects of interferon-alpha on serum hepatitis C virus in patients with chronic hepatitis C

Interferon is beneficial in some patients with chronic hepatitis C. To assess the efficacy of interferon, we used the polymerase chain reaction (PCR) to measure HCV RNA in serial serum samples from 13 chronic hepatitis C patients who were treated with interferon-alpha. Serum alanine aminotransferase (ALT) values normalized in association with the disappearance of serum HCV RNA in nine cases during the therapy. Serum HCV remained negative after the therapy in the three patients who had no relapse, while serum HCV RNA reappeared in the six patients with elevation of ALT values. The persistence of normal ALT levels appears to be correlated with the clearance of the serum HCV. There were two patients whose ALT became normal immediately after the cessation of interferon. Serum HCV was detectable at the end of treatment when serum ALT was elevated, and thereafter serum HCV disappeared. This result suggests an immunomodulatory effect of interferon in the clearance of HCV in some cases. Furthermore, the semiquantitative PCR assay showed that all five patients in whom ALT values were normal at the end of follow-up without detectable serum HCV genome had lower HCV titers in the pretreatment sera than the other eight patients. The detection of HCV RNA by the PCR assay is useful in determining the efficacy of interferon and its mechanisms.     

Clinical implications of GBV-C/HGV infection in patients with "HCV-related" chronic hepatitis

BACKGROUND/AIMS: To evaluate the clinical, biochemical and histological implications of a concomitant HGV infection in "HCV-related" chronic liver disease. METHODS: Eighty-three HCV-RNA positive patients with chronic liver disease were tested for GBV-C/HGV coinfection by heminested PCR. RESULTS: Twenty-two (26.5%) patients were found to be positive for GBV-C/HGV RNA. GBV-C/HGV+ patients differed significantly from GBV-C/HGV- ones for younger age, higher frequency of history of drug addiction, which in turn might favor coinfection with interferon-sensitive HCV genotypes (3a), and increased probability of long-term response to interferon. GBV-C/HGV infection appears to have no responsibility for specific aspects of HCV infection such as biochemical or histological cholestatic features, lymphoid follicles, symptomatic cryoglobulinemia or presence of serum autoantibodies, including LKM1. It does not worsen the HCV-related disease (ALT levels and histological activity) and does not significantly interfere with HCV infection, as explored by the number of hepatocytes positive for HCV antigens. The amount of steatosis (mean score) was shown to be higher in GBV-C/HGV+ patients. A virological follow up was performed in 17 interferon-treated GBV-C/HGV+ patients On the whole, GBV-C/HGV seems to be as sensitive to IFN treatment as HCV, but recurrence after withdrawal is more frequent. In spite of this, ALT levels often remain normal after treatment withdrawal. CONCLUSIONS: The present data suggest that GBV-C/HGV infection, apart from more marked liver steatosis, does not modify the overall picture of chronic hepatitis due to HCV infection.     

Hepatitis C virus genotype in anti-HCV-positive hemodialysed patients

We investigated the influence of hepatitis C virus (HCV) genotypes on the clinical course of HCV infection in a haemodialysis population. In June 1991, a 4 year prospective follow-up programme was implemented in 184 consecutive haemodialysis patients. Alanine aminotransferase (ALT) and gamma glutamine transferase (GGT) were performed every 2 months. When HCV antibody (Ab) (by second-generation ELISA) was positive, it was confirmed by RIBA 2 and HCV RNA amplification by PCR. The pattern of nucleotide sequence variability in the 5' non-coding region was categorized according to Simmonds' genotype classification. Risk factors including blood transfusions were evaluated. The levels of hepatic enzymes in HCV Ab-positive patients were retrospectively studied over a mean period of 11.8 years. ALT and GGT levels were assigned a score for every year of infection (0 = normal, 1 = fluctuating 2 = high levels). Fifty-two patients were HCV Ab reactive (30.4%), eight were RIBA undetermined and 44 were RIBA positive; 40 of these were HCV RNA positive (91%). Twelve patients were HCV RNA negative, suggesting that they had recovered from the infection. Four genotypes were identified: 1b [26 patients (65%)], 1a (one patient), 2 [12 patients (30%)] and 3 (one patient). The genotype distribution was not different from that found in patients with chronic hepatitis C and normal renal function of the same geographical area. Genotype 1b accounted for 75% of the cases before 1985 and an equal prevalence of the two major genotypes was observed after 1985. Patients infected with HCV subtype 1 had normal mean ALT levels, but higher levels in the follow-up period (28 +/- 15.6 IU/l) and higher ALT and GGT personal scores in the retrospective study. Genotype 1 patients had higher mean ALT levels after 6 months. HCV RNA-negative patients had lower ALT levels after 24 months. RIBA pattern could differentiate the patients. Patients with genotype 1 received a higher number of transfusions, while only 50% of HCV RNA-negative patients had been transfused. Our data suggest a worse course of HCV infection in haemodialysis patients infected with HCV subtype 1, but the severity of HCV infection can only be assessed by histology. Transaminases are only loosely correlated with severity.     

Effectiveness of relative low-dose interferon treatment for patients with chronic hepatitis C

To demonstrate effectiveness by relative low-dose of interferon treatment for patients with chronic hepatitis C, we investigated the histological activity index (HAI) score of liver tissue, hepatitis C virus (HCV) genotype by polymerase chain reaction (PCR) with type-specific primers, HCV RNA levels by competitive PCR, serum aminotransferase, sex, age, history of blood transfusion and history of hepatitis in patients with chronic hepatitis C prior to treatment. Twenty-two patients were treated with human lymphoblastoid interferon (3 MU daily, 2 weeks, 3 MU thrice a week, 6 weeks, 1.5 MU thrice a week, 16 weeks) for 24 weeks, of whom 10 (45.5%) were responders within a 6 month follow-up. HAI score before treatment was significantly lower in responders than in a combined group of relapse patients and nonresponders (7.5 +/- 3.4 vs. 12.0 +/- 3.1, p < 0.01). The prevalence of responders in patients with genotypes III and IV was significantly higher than in those with genotype II (85.7% vs. 21.4%, p < 0.05). HCV RNA level (logarithmic transformed copy per 50 microliter of serum) was significantly lower in responders than in a combined group of relapse patients and nonresponders (4.8 +/- 1.2 vs. 5.7 +/- 0.8, p < 0.05). In case of other pretreatment factors, there were no significant differences between responders and a combined group relapse patients and nonresponders. Thus severe histological changes in the liver, HCV genotype II and high HCV RNA levels are markers of unfavorable effects of interferon treatment for patients with chronic hepatitis C.     

Quantitation of hepatitis G and C viruses in the liver: evidence that hepatitis G virus is not hepatotropic

Hepatitis G virus (HGV) is prevalent in patients with chronic liver disease and has been previously detected in liver specimens. However, it is unknown whether the virus is replicating in the liver or is simply a contaminant from serum. We sought to determine whether HGV was hepatotropic and to determine whether coinfection with HGV and hepatitis C virus (HCV) influenced the level of either virus. Virus was quantitated using branched DNA (bDNA) assay for both HGV and HCV in the liver explants and pretransplant serum samples from 30 transplant recipients: Group I, HGV/HCV coinfection (n = 10); group II, HCV infection alone, (n = 8); group III, HGV alone (n = 12). In patients with coinfection HCV (RNA) titers in liver were consistently higher than those for HGV RNA (median 1.13 x 10(8) and 360,000 Eq/g respectively, P < .01). The ratio of liver/serum viral RNA was significantly higher for HCV than for HGV (median 129 and 0.3 respectively, P < .01). Levels of HCV RNA were similar in patients with HCV infection alone versus those with HGV/HCV coinfection (median; liver = 1.15 x 10(7) vs. 1.13 x 10(8) Eq/g, serum = 500,000 vs. 200,000 Eq/mL) and levels of HGV RNA in liver and serum were similar in patients with HGV infection alone compared to those with HGV/HCV coinfection (median; liver = 1.2 x 10(6) vs. 4.0 x 10(5) Eq/g, serum = 4.5 x 106 vs. 2.6 x 10(6) Eq/mL). Levels of either virus appeared unaffected by the presence of an additional virus. The high ratio of HCV RNA levels in liver compared to serum is consistent with its known hepatotropism, but this pattern was not observed for HGV. The median liver/serum ratio of HGV RNA was less than unity, a finding consistent with serum contamination of liver tissue. Thus we conclude that the liver is not the main site of HGV replication.     

High-dose (9 MU) long-term (60 weeks) alfa-interferon therapy for chronic hepatitis patients infected with HCV genotype 1b

Efficacy of standard regimens (e.g., 3-6 MU for 24 weeks) of alfa-IFN therapy for chronic hepatitis C has been limited, particularly in patients with HCV/1b. To see if higher-dose longer term treatment is more effective, we tried a 9 MU 60-week regimen. HCV/1b-infected chronic hepatitis patients received 9 MU IFN alpha 2a everyday but Sunday for 2 weeks and thrice a week for next 10 weeks, and 76 patients became HCV RNA-negative while 81 remained positive. The RNA-negative patients were then randomized to receive 3 MU (group I, n = 37) or 9 MU (group II, n = 39) for 48 weeks. Of the RNA-positive patients, only those with normal ALT received another 9 MU 48-week treatment (group III, n = 45). Sustained responders (SR) were defined as those with negative RNA and normal ALT 6 months after the therapy. SR rates based on intent-to-treat principle did not differ significantly between groups I and II (30% vs 41%), but those based on the protocol-compatible cases showed a significantly lower than those in group II. Adverse effects of IFN, developed more frequently in groups II and III than in group I, were mostly reversible. In conclusion, our results encourage 9 MU 60-week IFN alpha treatment in HCV/1b-infected patients with careful attention to adverse effects, and suggest that the treatment should be discontinued if HCV RNA does not disappear within 12 weeks.     

Disseminated miliary tuberculosis of the skin in patients with AIDS: report of four cases

During the course of long-term follow-up, we examined the efficacy of interferon (IFN) in the improvement of liver function and prevention of hepatocellular carcinoma (HCC) in hepatitis C virus (HCV) associated cirrhosis patients. Fifty-five cirrhotic patients, in whom HCC nodules in the liver were not detected by ultrasonography (US) or computed tomography (CT), received 3 or 6 million units of human lymphoblastoid IFN daily for two weeks and 3 times a week for 22 weeks. Complete response (CR) was defined as normalization of serum alanine aminotransferase (ALT) together with negative HCV RNA at 6 months after IFN therapy completion. Any other pattern of response was defined as non-response (NR). After IFN therapy the patients were followed up every 1-3 months for at least 1 year (average follow-up period, about 40 months) with serological tests and US or CT. In the 8 CR patients, the serum ALT levels remained normal and HCV RNA remained negative. Platelet count, white blood cell count, serum albumin and zinc turbidity test have recovered to the normal range at final follow-up. Ten of the 47 patients with NR have developed HCC, whereas no patients with CR has developed HCC during follow-up. We conclude that IFN improves the liver function and may prevent the development of HCC even in cirrhotic patients who show CR to IFN therapy.