Toyomi Matsumoto

Genotypic subtyping of hepatitis C virus

Four subtypes of hepatitis C virus (HCV), Pt(I), K1(II), K2a(III) and K2b(IV), have been suggested based on the nucleotide sequences of the non‐structural (NS) 5 region. A fifth subtype from Japanese patients, Tr(V), which shows a less than 68% homology in nucleotide sequence when compared with other subtypes has been identified. A one‐step method which enables a quick determination of subtype using polymerase chain reaction with a mixed primer set deduced from the sequence of each subtype has been developed. Using this technique, the subtypes of 418 out of 478 Japanese patients (87.4%) were determined. The incidence of each subtype in Japan was as follows: K1(II), 307 (73.4%); K2a(III), 74 (17.7%); K2b(IV), 28 (6.7%); and Tr(V), 3 (0.7%). This one‐step subtyping technique should be useful for studying the epidemiology or biology of the HCV.

Factors useful in predicting the response to interferon therapy in chronic hepatitis C

To determine how various factors influence the response to interferon (IFN) therapy, we retrospectively studied 157 consecutive Japanese patients with chronic hepatitis C who received various treatment schedules of IFN. They were divided into two groups on the bases of outcome. One group was comprised of 65 patients who achieved a sustained normalization of serum alanine aminotransferase (ALT) levels for at least 6 months after treatment, while the other group was comprised of 84 patients with persistent elevation of serum ALT levels, despite treatment. Genotyping of hepatitis C virus (HCV) was done by polymerase chain reaction (PCR) with genotype specific primers, analysing the variations in nucleotide sequence within the NS 5 region of the HCV genome, namely genotypes PT, K1, K2a and K2b. We then used a multivariate analysis to determine the factors related to mode of treatment, patient characteristics and HCV genotype in relation to the response to IFN therapy. Of the 16 factors analysed, the HCV genotype (genotype K2a or K2b, P < 0.0008), treatment schedule (intermittent administration following a daily schedule, designated as combined schedule, P > 0.0014) and liver histology just before treatment (chronic persistent hepatitis or mild chronic aggressive hepatitis, P < 0.0324) were the most strongly correlated with a normalizing response to IFN therapy. These results suggest that not only are the IFN treatment schedule and patient profile significant, but the properties of the virus also influences the response. However, as the IFN treatment schedule is the only changeable factor, it should be designed to maximize the benefit of IFN therapy.

Quantitative analysis of hepatitis C virus RNA by competitive nested polymerase chain reaction

A method that allows the quantitation of hepatitis C virus (HCV) RNA is described. The RNA was extracted from serum samples and reverse transcribed. Target cDNA was then co‐amplified by nested polymerase chain reaction with a known amount of competitive template at various concentrations. Since this internal control DNA uses the same primers as those of the target and is distinguishable from the target cDNA after amplification by size, the initial concentration of the target could be estimated by comparing the intensity of the two bands of amplified DNA fragments. That is, if the starting amount of the cDNA and the competitive template are equal, the intensity of the two bands should also be equal. Using this method the amount of HCV RNA in serum samples obtained from 85 patients with chronic hepatitis type C was determined. There was as much as a 100 000‐fold difference in the levels of HCV RNA from patient to patient. A rapid decrease of HCV RNA in a patient treated with interferon is also described.

Genotype, slow decrease in virus titer during interferon treatment and high degree of sequence variability of hypervariable region are indicative of poor response to interferon treatment in patients with chronic hepatitis type C

In a study assessing factors associated with a good or a poor response to interferon treatment in patients with chronic hepatitis type C, we analyzed serum samples taken from 26 interferon-treated patients and found further evidence that infection with genotype II is associated with a poor response. Whereas all seven patients with group III genotype tested showed a good response (normalization of alanine aminotransferase level for more than 6 months), only 10 of 19 (53%) patients infected with group II genotype showed a good response. We also observed that 16 of 17 (94%) patients who exhibited a rapid virus titer decrease during the first 2 weeks of treatment later showed a good response. In contrast, only three of nine (33%) patients with an initially slow viral decrease eventually showed a good response (p<0.04). None of the 26 control patients exhibited a marked virus decrease or normalization of serum alanine aminotransferase level. Interestingly, high degrees of sequence variability were seen in three patients with group II hepatitis C virus who responded poorly to the therapy. All three showed slow decreases in virus titer during the first 2 weeks of treatment. In contrast, patients with genotype II who showed a good response to treatment were seen to have very few mutations. In three patients with genotype III who had responded well to interferon treatment, all showed very little amino acid sequence variability in the hypervariable region compared with patients with genotype II who had responded poorly to interferon treatment. These data suggest that a slow decrease in virus titer during the beginning of interferon treatment and a high degree of sequence variability, both of which are often seen in patients with group II genotype, are associated with poor response to interferon treatment.

Interferon retreatment of nonresponders with HCV-RNA-positive chronic hepatitis C.

Interferon has been shown to be an effective treatment for some patients with chronic hepatitis C. In this study, the value of retreatment of nonresponders to interferon was investigated. Thirty-eight patients with hepatitis C virus (HCV)-RNA-positive chronic hepatitis C virus (HCV)-RNA-positive chronic hepatitis C who had been treated with betainterferon but still showed an alanine aminotransferase (ALT) level>50 KU upon completion of therapy were retreated with alpha-interferon. Eight patients (21.1%) had normalization of ALT levels after interferon retreatment were studied. Of 16 patients with transient HCV-RNA negativity 1 month after the initial interferon therapy, 7 (43.8%) had a complete response, with normalization of ALT levels and undetectable HCV-RNA, more than 6 months after interferon retreatment. On the other hand, of the 22 patients with HCV-RNA activity 1 month after the initial interferon therapy, only 1 (4.5%) had a complete response. Multivariate analysis, using a multiple logistic model, indicated that a complete response to readministration of interferon was most strongly correlated to transient negative conversion for HCV-RNA after the initial course of treatment.

Antiviral effect of lymphoblastoid interferon‐alpha on hepatitis C virus in patients with chronic hepatitis type C

Abstract To study the antiviral effect of lymphoblastoid alpha interferon (IFN) on hepatitis C virus (HCV) we conducted a randomized, controlled trial on 80 patients with chronic hepatitis C using three different doses. Patients were randomly assigned to treatment with 1, 3 or 6 million units of lymphoblastoid IFN‐alpha daily for 2 weeks. To assess the antiviral effect of IFN, the amount of HCV present in the serum was estimated by competitive nested polymerase chain reaction (PCR) before and after 2 weeks of treatment. The multiple logistic analysis was used to evaluate factors associated with virus clearance, adjusting the imbalance in predictive factors among patients. Hepatitis C virus became negative as assessed by nested PCR after therapy in 26, 50 and 63% of patients receiving 1, 3 and 6 mega units, respectively. Hepatitis C virus was cleared more often in patients having initially low (< 105/mL) amounts of virus. No significant decrease in the amount of virus was observed in the untreated, control group. Patients without bridging fibrosis in liver histology and with HCV genotypes other than K1 (type II) tended to respond well. These results indicate that lymphoblastoid IFN‐alpha suppresses HCV in a dose dependent manner. Higher initial virus amounts, bridging fibrosis and genotype K1 were factors associated with poor response.

Effect of lymphoblastoid alfa-interferon in patients with chronic hepatitis C having different genotypic subtype of hepatitis C virus

SummaryFive subtypes of hepatitis C virus (Pt[I], K1[II], K2a[III], K2b[IV] and Tr[V]) have been suggested based on the nucleotide sequence of the non-structural five region. To assess the susceptibility of each subtype to interferon therapy, we developed a one-step method which allows quick determination of subtype using polymerase chain reaction with a mixed primer set deduced from the sequence of each subtype. We were able to determine the subtype of 69 of 80 (86.3%) Japanese patients who received natural alfa-interferon treatment. The incidence of each subtype was K1: 53 (76.8%), K2a: 14 (20.3%), K2b: 3 (4.3%) and Tr: 1 (1.4%). Interferon was administered to these patients and found to be effective in 28 of 51 (54.9%) patients with K1 subtype and in 16 of 18 (88.9%) patients with other subtypes (P< 0.01). These data show that K1 is a major subtype in Japan and relatively resistant to interferon treatment.

Alanine aminotransferase and HCV-RNA responses following interferon therapy of HCV-RNA positive chronic hepatitis.

SummaryInterferon (IFN) has been shown to be effective for chronic hepatitis C. This study investigated changes of alanine aminotransferase (ALT) and HCV-RNA in chronic hepatitis C patients treated withα-IFN. IFN was given to 73 patients with HCV-RNA positive chronic hepatitis C. The pattern of changes in ALT activity after IFN aministration was classified into five types. Type 1 was characterized by normalization of ALT (≤25 K.U) during IFN administration and sustained normalization after the IFN therapy. Type 2 involved a rebound of ALT after termination of IFN therapy and subsequent normalization. Type 3 had no ALT normalization during IFN administration, with normalization after the completion of the therapy. Type 4 involved transient normalization of ALT level during IFN therapy, with a subsequent reversion to abnormal levels after the termination of IFN therapy. Type 5 showed sustained abnormally high levels of ALT activity both during and after treatment. Twenty four patients (32.9%) had sustained normalization ALT (≤25 K.U) after the termination of IFN treatment. The HCV-RNA negative rate at 6 months after IFN therapy in patients with sustained normalization of ALT was 87.5% (21/24).