Hiromi Koike

Susceptibility of TT virus to interferon therapy.

TT virus (TTV) is a newly identified single-stranded DNA virus. We retrospectively analysed serum samples from sixteen patients, infected with both hepatitis C virus (HCV) and TTV, and who had been treated with interferon. An elevated serum alanine aminotransferase level after interferon was associated with persistence of HCV (abnormal in five of seven patients with persistence of HCV compared with normal in all nine patients who showed eradication of HCV) irrespective of persistence of TTV. Comparison of partial viral DNA nucleotide sequences and phylogenetic analysis showed that viral strains that had a high identity to the prototype virus were more resistant to interferon than those showing low nucleotide sequence identity. Although we observed no liver cell injury caused by persistent TTV infection, the mechanism(s) of TTV resistance to interferon should be further investigated for a better understanding of viral diseases and establishment of therapy.

Prevalence of TT virus before and after blood transfusion in patients with chronic liver disease treated surgically for hepatocellular carcinoma

Background: To examine the prevalence of TT virus (TTV) before and after blood transfusion, we retrospectively examined serum samples obtained from 55 patients who received blood transfusions before, during and after resection of hepatocellular carcinoma.

Fluctuations of hepatitis C virus load are not related to amino acid substitutions in hypervariable region 1 and interferon sensitivity determining region

Hepatitis C virus (HCV) load is one of the most important predictive factors of response to interferon treatment. However, little is known about the mode and determinants of viremia. The mode of viremia was investigated in 78 patients with chronic HCV genotype 1b infection during 1–2 years follow up. Virus load, determined by a branched chain DNA amplification assay, was stable in 73 of 78 (93.6%) patients, whereas 5 (6.4%) showed marked fluctuation (from undetectable level to more than 10 Meq/ml) in viral titer. To study the mechanisms mediating fluctuations in viral titer, amino acid sequences of two regions were examined; hypervariable region (HVR) 1 and the interferon sensitivity determining region (ISDR). Multiple amino acid substitutions were observed in HVR 1 but no relationship was evident between substitutions and virus titers. In contrast, no amino acid substitutions were observed in the ISDR in any patients with stable virus titer during a follow‐up period of 12–24 months (7–24 samples) or in one patient who was observed for 15 years. Interestingly, multiple amino acid substitutions in the ISDR appeared in only two of the five patients with marked titer fluctuation, when the virus decreased markedly. Alanine aminotransferase levels in these five patients correlated with viral load. The data suggest that amino acid substitutions in HVR1 and ISDR are not essential for changes in viral titer. Possible mechanisms of fluctuations of viral titer and amino acid substitutions in the ISDR accompanying marked reductions in viral load are discussed. J. Med. Virol. 58:247–255, 1999.

Typing six major hepatitis C virus genotypes by polymerase chain reaction using primers derived from nucleotide sequences of the NS5 region

Abstract We recently developed a polymerase chain reaction (PCR) based method for determining hepatitis C virus (HCV) genotypes using primers derived from nucleotide sequences of the NS5 region. Using this method, we determined 5 hepatitis C virus (HCV) genotypes (1a, 1b, 2a, 2b and 3b) detected in Japanese patients. Following the recent identification of genotype 3a HCV in a patient from Pakistan currently living in Japan, we improved the typing method used for detecting all of these 6 genotypes. Eighteen of 1100 HCV RNA positive patients who tested negative for genotype by the original method also tested negative by the new method. Type 3a genotype HCV was thus shown to be very rare among Japanese patients with HCV infection. Nucleotide sequence and quantitative analysis of these negative samples revealed that negative results were due to mutations present on the nucleotide sequence where the 3′ end of the primers was situated due to a very low titer (less than 103) of the virus. The new single step genotyping method may be clinically useful as it allows a fast determination of all 6 genotypes present in Japan.

Enzyme-linked immunosorbent assay to detect hepatitis C virus serological groups 1 to 6.

Abstract: By conventional serological grouping methods, it is possible to determine hepatitis C virus (HCV) serological groups for genotypes 1a, and 1b, and genotypes 2a, and 2b, but not for other genotypes, i.e., 3a, 3b, 4a, 5a, and 6a. In this study, we attempted to serologically group HCV with the Murex HCV serotyping 1 to 6 assay (Murex Diagnostics, Kent, UK), using an enzyme-linked immunosorbent assay (ELISA) based on genotype-specific peptides from the NS4 region. The subjects of this study were 365 patients infected with HCV of genotype 1a, 1b, 2a, 2b, 3a, or 3b. The sensitivity of the assay was 100% in patients with genotype 1a, 82.7% in those with 1b, 68.5% in those with 2a, 84.2% in those with 2b, 50.0% in those with 3a, and 76.5% in those with genotype 3b. The overall sensitivity was 78.4%. The specificity of the assay was 100% in the subjects with genotype 1a, 98.8% in those with 1b, 98.4% in those with 2a, 96.9% in those with 2b, 100% in those with 3a, and 100% in those with genotype 3b. The overall specificity was 98.6%. The concordance of the assay was 100% in subjects with genotype 1a, 81.7% in those with 1b, 67.4% in those with 2a, 81.6% in those with 2b, 50.0% in those with 3a, and 76.5% in those with genotype 3b. The overall concordance was 77.5%. We believe it would be better to serotype with the Murex HCV serotyping 1 to 6 assay, if other than serological group (Gr) 1 or Gr 2 is suspected in particular ethnic groups or in subjects with an indeterminate result with the Immucheck HCV Gr assay (Kokusai, Kobe, Japan), assuming that the genotype must be other than 1a, 1b, 2a, or 2b.

Predictive factors in eradicating hepatitis C virus using a relatively small dose of interferon.

Interferon (IFN) can reduce hepatitis C virus load and even eliminate the virus in 30‐40% of patients. Several predictive factors for eradication of the virus have been reported and a higher dose of IFN tends to result in elimination of the virus. However, a small dose of IFN sometimes is as effective as a large dose in eradicating the virus. The predictive factors for such a response are not well established. We retrospectively analysed 50 patients with chronic hepatitis C who were treated with relatively small amounts of IFN (equal or less than 252 million units). Eleven patients were responders (elimination of hepatitis C virus (HCV) and normalization of alanine amino transferase (ALT) for at least 6 months), but the remaining 39 were non‐responders. Multivariate analysis showed that the pretreatment viral load and total dose of IFN per kilogram of bodyweight were significant predictive factors of response to therapy. We also assessed the amino acid substitutions in the IFN sensitivity determining region (ISDR), NS5A codon 2209‐2248, of HCV in serum samples obtained from 31 patients with HCV genotype 1 b. The presence of more than one amino acid substitution in the ISDR tended to correlate with HCV genotype lb elimination. As IFN is expensive and has a number of serious side effects, our study suggests that the optimal dose of IFN may vary from one patient to another and that more stringent criteria should be used to select the optimal dose for therapy.

Predictive value of different hepatitis C serological assays in the treatment of chronic hepatitis C with interferon α

Abstract: In order to predict the complete response rate of natural interferon-α (nIFN-α) treatment in patients with chronic active hepatitis C, we examined the predictive value (PV) of different hepatitis C serological assays. We performed first generation (ver.1) and second generation (ver.2) hepatitis C virus (HCV) branched DNA-probe assays (bDNA-probe), HCV core protein assay (core protein), HCV Amplicor Monitor assay (amplicor monitor), and HCV competitive polymerase chain reaction (competitive PCR) assay, using serum samples collected immediately before initiation of treatment. For each marker, we studied, in patients stratified by serological group (Gr), which predictive value (PV) of the HCV titers showed association with the therapeutic effect. In 59 Gr 1 patients, complete response to nIFN-α treatment was predicted from the following PVs for each marker: 0.5 Meq/ml or less (odds ratio 11.7; P = 0.0010) with ver.1, 1.0 Meq/ml or less (odds ratio 5.3; P = 0.0119) with ver.2 of the bDNA-probe, 50 pg/ml or less (odds ratio 10.3; P = 0.0062) with core protein, 200 × 103 copy/ml or less (odds ratio 7.8; P = 0.0031) with amplicor monitor, and 104 copy/ml or less (odds ratio 6.2; p = 0.8395) with competitive PCR. In 27 Gr 2 patients, the PV for each marker indicating complete response was as follows: There was no relationship between PV and therapeutic effect with ver.1 of the bDNA-probe, while the PVs for the other markers were 0.2 Meq/ml or less (odds ratio 2.2; P = 0.3788) with ver.2, 20 pg/ml or less (odds ratio 5.6; P = 0.0597) with core protein, 400 × 103 copy/ml or less (odds ratio 4.0; P = 0.2965) with amplicor monitor, and 105.5 copy/ml or less (odds ratio 29.2; P = 0.0096) with competitive PCR. Our findings showed that complete response to the treatment may be predicted using the appropriate PV for each marker.

Nucleotide sequences of hepatitis GB virus C. Identification of highly conserved domains in the 5' noncoding region and detection by polymerase chain reaction

Abstract We detected the hepatitis GB virus C genome by reverse transcription-polymerase chain reaction in three Japanese patients with chronic liver diseases. Partial nucleotide sequences of 5′ noncoding, envelope and NS3 regions had 88.6–90.1, 86.388.0, and 78.6–79.6% nucleotide sequence homology compared with the prototype GBV-C genome. However, they showed higher homology with each other (96.197.1, 88.7–91.2, 84.0–87.0%, respectively), suggesting that they were a genotype of GBV-C. The domains that were highly conserved among all these genomes were present in the 5′ noncoding region. The frequency of detecting the genome by the polymerase chain reaction was higher when we used primers designed on these domains compared with primers designed on the core and NS3 region. The virus genome was detected in eight of 50 (16.0%) consecutive Japanese patients with hepatocellular carcinoma who had received more than ten units of blood transfusion, three of 60 (5.0%) patients with non-B, non-C liver disease and 12 of 35 (34.3%) Malaysian patients with non-B chronic liver disease. Since there are no reliable assays to detect hepatitis GB virus C at present, the detection of the genome by the polymerase chain reaction should be useful for diagnosis. Further nucleotide sequence analysis of the genomes is necessary for epidemiological survey studies and vaccine strategy.

Biochemical and histological features of hepatitis G virus infection

To assess the biochemical and histological characteristics of hepatitis G virus (HGV) infection, we examined four patients who were infected with HGV only (HGV group), and compared them with 16 patients infected with both HGV and hepatitis C virus (HCV; HGV + HCV group) and 18 patients infected with HCV only (HCV group). Biochemical examination showed a significantly low level of serum alanine aminotransferase (ALT) in the HGV group, and that the gamma‐glutamyl transpeptidase (γ‐GTP)/ALT ratio in the same group was significantly higher than in the other two groups. Although all three patient groups had a similar degree of liver fibrosis, both the degree of periportal inflammation and total histological activity index were significantly lower in the HGV group than in the other two groups. Fibrous enlargement of the portal tract without lymphoid infiltration and thin fibrous septa was characteristically observed in the HGV group. No significant difference was found between the HGV + HCV group and HCV group. Our results suggest that biochemical and histological changes in HGV infection are very mild and quite different from those of HCV infection.

Genotyping of hepatitis C virus by the polymerase chain reaction: a comparison of two PCR-based methods using core or NS5 primers

Abstract The presence of hepatitis C virus subtypes and their clinical importance, especially in the context of interferon sensitivity, are reported. In this study, we compared two kinds of polymerase chain reaction (PCR)-based methods—one step quick subtyping and two stage subtyping procedure for the determination of genotypes. Using the mixed primer sets derived from the core or NS5 region, we amplified cDNA samples obtained from patients with a hepatitis C virus infection. Of 116 samples tested, 91 (78%) showed different ones. Although such discrepant results are considered to be related to the characteristics of these two methods, further studies remain to elucidated.