Polymerase chain reaction (PCR) and the new branched-DNA method for detecting HCV RNA have greatly facilitated the assessment of specific treatment efficacy [3]. These techniques can be used to detect ongoing HCV replication and the persistence of HCV genomes in the liver and peripheral blood mononuclear cells [4]. However, they have not yet been used to evaluate hepatic, serum, and peripheral blood mononuclear cell-HCV RNA status in long-term responders to -interferon.

We evaluate whether -interferon therapy leads to complete eradication of the virus in some long-term responders because the HCV genome does not integrate host-cell DNA, and its persistence thus depends on HCV RNA replication.

Methods

Top Methods Results Discussion Author & Article Info References

Between January 1990 and March 1993, 348 anti-HCV-positive patients were referred to our hepatology unit. The 189 patients who were given -interferon therapy met the following criteria: hypertransaminasemia, detectable HCV viremia, and biopsy-proven chronic hepatitis. The 159 patients who were not treated had either a relative contraindication (for example, renal transplantation, hemodialysis, human immunodeficiency virus [HIV]-associated infection, chronic alcoholism, thyroid disease, or normal aminotransferase activity) for such a treatment or had mild liver disease; other patients refused -interferon treatment or liver biopsy.

Sufficient follow-up was available for 103 of the 189 patients so that long-term response to treatment could be assessed. Eighty-five patients were followed for less than 6 months after withdrawal of -interferon, which precluded their inclusion in the study. Twenty-one of the 103 treated patients (20.4%) were long-term responders (defined by normal aminotransferase levels at least 6 months after the end of treatment). Our study is based on the 9 of 21 patients whose serum, liver, and peripheral blood mononuclear cells samples were available for HCV RNA detection.

These nine patients included five men and four women with a mean age of 33 years (range, 25 to 62 years) whose risk factors for viral infections were previous intravenous drug addiction (five patients), blood transfusion (one patient), and unknown (three patients) (Table 1). All had anti-HCV antibodies as shown by second-generation enzyme-linked immunosorbent assay or recombinant immunoblot assay. Before therapy, eight patients had biopsy-proven chronic active hepatitis, and one had biopsy-proven cirrhosis. They were treated with 3 mU of -2b-interferon subcutaneously 3 times a week for 6 months (six patients) or 12 months (three patients). They were monitored for a mean period of 17.5 months (range, 6 to 36 months), and a liver biopsy was done at the end of this period in all but one case: For patient 4, a liver biopsy was obtained 8 months before the end of follow-up and 6 months after -interferon was withdrawn (Table 1). Frozen serum samples taken before and after treatment were available in every case. Frozen liver samples taken before treatment (n = 6) and after treatment (n = 7) were also available. Peripheral blood mononuclear cells were obtained from five patients after treatment.

View this table: [in this window] [in a new window]   Table 1. Virologic and Histopathologic Findings before and after -Interferon Treatment*

Polymerase chain reaction was done using primers located in the conserved 5' noncoding region of the HCV genome. The sensitivity of our assay for HCV RNA has been assessed by use of a reference panel [5]; we are now able to detect HCV RNA in 10^–7 dilutions of a chimpanzee-infective dose. The assay can also detect the RNA of approximately 50 HCV particles per µL of serum [4]. Primers specific for cyclin A were used to test whether the quality of the purified cellular RNA was adequate for PCR. Genotyping was done according to the Okamoto technique [6]. Polymerase chain reaction was done with one universal and five type-specific primers. Type-specific PCR products were clearly recognized by their distinct size after they had been subjected to electrophoresis and stained with ethidium bromide.

Results

Top Methods Results Discussion Author & Article Info References

All patients were positive for serum HCV RNA before treatment; HCV RNA was also detected in the six frozen liver specimens that were obtained before treatment. Genotype 1b (II) was detected in one of seven treated patients, and genotypes 3a and 1a were detected in four and one patients, respectively. One sample could not be classified.

After treatment and at the end of follow-up, no patients remained positive for serum HCV RNA. None of the five peripheral blood mononuclear cell samples and only one of the seven frozen liver samples contained detectable HCV RNA (Table 1). The nine liver biopsy specimens obtained after treatment showed a pattern consistent with minimal histologic liver change in seven patients, disappearance of the signs of histologic activity (necrosis and inflammation) in the patient with cirrhosis, and chronic persistent hepatitis in one patient. Minimal histologic changes occurred in the patient whose liver sample remained HCV RNA-positive.

Discussion

Top Methods Results Discussion Author & Article Info References

Our findings suggest that -interferon therapy can lead to complete eradication of HCV infection in long-term responders. This is manifested by a complete and sustained normalization of aminotransferase activity after stopping treatment. Hepatitis C virus RNA was undetectable in the frozen liver, peripheral blood mononuclear cell, and serum samples from the patients concerned despite the use of a sensitive PCR method and strict quality controls to check the integrity of the nucleic acids extracted from the liver and peripheral blood mononuclear cells. The disappearance of HCV RNA from the liver was associated with a substantial decrease in liver-cell necrosis and inflammation. Several patients who had mild chronic active hepatitis before treatment showed only minimal histologic changes after treatment. We felt it important to test peripheral blood mononuclear cells because several studies have strongly suggested that these cells are a potential extra-hepatic site of HCV persistence [7]. The negative results we obtained from these samples are consistent with the results from the serum and liver samples and further support the hypothesis that the virus was completely eradicated from these patients. Similar observations have recently been described in Spanish patients [8]. Genotype 1b (II) was observed in only one of our long-term responders, a finding in accordance with those from other series [8, 9]. This reinforces the view that genotyping is helpful in prognosis. Despite the encouraging nature of these results, the study sample was small and highly selected; confirmation is necessary in a larger group of patients.

The virus, however, was not eliminated from all patients whose aminotransferase activity returned to normal in repeated samples. In one patient, HCV RNA was present in the liver even though it was undetectable in serum. Nonetheless, histologic examination of the liver after treatment showed a substantial improvement in this patient, with minimal histologic changes. This patient could be considered an asymptomatic HCV carrier [10], although some controversy exists about whether chronic HCV infection can be associated with an asymptomatic chronic carrier state [10, 11]. Our study suggests that testing for HCV RNA in the liver may be helpful when evaluating the persistence of HCV infection in patients with sustained normal alanine aminotransferase levels after treatment.