Hepatitis B surface antigen reduction by switching from long-term nucleoside/nucleotide analogue administration to pegylated interferon.

Hepatitis B surface antigen (HBsAg) reduction during nucleoside/nucleotide analogue (NA) therapy is slow and an alternative strategy for patients receiving ongoing NA to facilitate HBsAg reduction is required. We investigated whether switching to pegylated interferon (PEG-IFN) after long-term NA administration enhances HBsAg reduction. Forty-nine patients who switched from long-term NA to 48 weeks of PEG-IFN alfa-2a were studied. The mean duration of previous NA was 48 months (sequential group). A total of 147 patients who continued NA and matched for baseline characteristics were analysed for comparison (NA continuation group). The treatment response was defined as HBsAg reduction ≥1.0 logIU/mL at the end of PEG-IFN. HBsAg reduction at week 48 was 0.81±1.1 logIU/mL in the sequential group, which was significantly higher than that in the NA continuation group (0.11±0.3 logIU/mL, P < .001). The treatment response was achieved in 29% and 2% of the sequential group and NA continuation group (P < .001), and the odds ratio of sequential therapy for the treatment response was 19 compared with the NA continuation (P < .001). In patients tested positive for hepatitis B e antigen (HBeAg), HBeAg seroconversion was higher in the sequential group (44% vs 8%, P < .001). In HBeAg-negative patients, only patients in the sequential group achieved HBsAg loss. No patient needed to resume NA administration because of HBV DNA increase accompanied by alanine aminotransferase flares. In summary, sequential therapy with PEG-IFN after long-term NA enhances the reduction of HBsAg and may represent a treatment option to promote HBsAg loss.

ITPA genetic variants influence efficacy of PEG-IFN/RBV therapy in older patients infected with HCV genotype 1 and favourable IL28B type.

Inosine triphosphatase (ITPA) genetic variants are strongly associated with ribavirin (RBV)-induced anaemia during pegylated interferon (PEG-IFN) plus RBV therapy. However, the treatment efficacy of ITPA genetic variants has not been fully explored. We enrolled 309 individuals infected with hepatitis C virus genotype 1, who were treated with PEG-IFN plus RBV for 48 weeks. The ITPA SNP: rs1127354 and IL28B SNP: rs8099917 were genotyped. We examined the risk factors for severe anaemia up to week 12 after the start of treatment and treatment efficacy. The incidence of severe anaemia, ≥ 3 g/dL reduction or <10 g/dL of haemoglobin (Hb) up to week 12, was more frequent in patients with CC at rs1127354 [65% (145/224), 33% (73/224)] than in those with CA/AA [25% (21/85), 6% (8/85)] (P < 0.0001). ITPA genotype, pretreatment Hb level and age were independent predictive factors for severe anaemia: Hb < 10 g/dL. In IL28B favourable type, the sustained virologic response rate was higher in ≥ 60-year-old patients with CA/AA than in those with CC [71% (22/31) vs 40% (26/65), P = 0.005], although there was no significant difference in treatment efficacy according to ITPA genetic variants in the <60-year-old patients. The proportion of patients administered ≥ 80% of the dosage of RBV was significantly higher in the patients with CA/AA than in those with CC (P = 0.025), resulting in a lower relapse rate. In conclusion, ITPA genetic variants were associated with severe RBV-induced anaemia and could influence the efficacy of PEG-IFN plus RBV treatment among elderly patients with IL28B favourable type.

Preoperative drainage using a transanal tube enables elective laparoscopic colectomy for obstructive distal colorectal cancer.

BACKGROUND AND STUDY AIMS: Acute colorectal obstruction (ACO) often accompanies colorectal cancer (CRC) and requires urgent treatment, but achieving elective laparoscopy-assisted colectomy (LAC) is difficult in this setting. The aim of the current study was to assess the clinical outcomes of a transanal tube (Dennis colorectal tube [DCT]) for CRC with ACO, focusing in particular on the impact of the DCT on subsequent elective LAC. PATIENTS AND METHODS: Among 1142 patients who underwent surgery for CRC between January 2007 and December 2011, 92 patients with ACO were identified retrospectively. Of these 92 patients, the DCT procedure was performed in 66 patients who fulfilled the indications for DCT, and these patients were included in the study. RESULTS: All 66 patients presented with complete obstruction. Technical and clinical success rates for DCT were 93.9 % and 86.4 %, respectively. Perforation after DCT occurred in 4.5 % and the mortality rate was 1.5 %. The rate of LAC was 48.5 %, and the rate of primary stoma was 13.6 %. For curative stage II/III CRC with ACO, DCT resulted in a primary stoma rate of 13.6 %, a one-stage surgery rate of 90.9 %, a LAC rate of 50.0 %, and a 3-year survival rate of 73.1 %. For stage II/III CRC cases with clinical success by DCT, the one-stage surgery rate was 97.4 % and the LAC rate was 56.4 %. CONCLUSIONS: DCT achieved a high rate of clinical success and enabled safe one-stage surgery and LAC for CRC with ACO. DCT followed by LAC is proposed as a promising non-invasive strategy for CRC with ACO.

Possible prediction of adverse reactions to pyrimidine chemotherapy from urinary pyrimidine levels and a case of asymptomatic adult dihydropyrimidinuria.

Deficiency of dihydropyrimidine dehydrogenase or dihydropyrimidinase, enzymes that catalyze the breakdown of pyrimidine chemotherapy agents such as 5-fluorouracil, may cause serious adverse reactions to these agents. We attempted to establish the reference range for urinary pyrimidines in adults to detect individuals with abnormal pyrimidine metabolism. We analyzed urinary pyrimidine levels in 1133 adults to establish a reference range for persons ages 20 years or older. Urinary dihydrouracil and uracil levels were determined by high-performance liquid chromatography with column switching. The reference range obtained was found to be 0-59.3 micromol/g creatinine for dihydrouracil and 0-129.8 micromol/g creatinine for uracil. In addition, an asymptomatic man with suspected dihydropyrimidinase deficiency was detected on the basis of dihydropyrimidinuria. Although only three cases of this disease have been found worldwide, including one infant reported previously by our group, it may not be so rare as has been thought. In this man, a 10 mg/kg oral uracil loading test yielded a peak blood dihydrouracil level of 192.1 micromol/liter and a peak uracil level of 67.8 micromol/liter. Eight h after loading, the uracil level was still 11.1 micromol/liter, about 17 times that in healthy subjects. Additional research on dihydropyrimininase deficiency may help to prevent adverse reactions to pyrimidine chemotherapy agents in susceptible individuals.

Evolutionary relationship of hepatitis C, pesti-, flavi-, plantviruses, and newly discovered GB hepatitis agents.

Two flavivirus-like viruses, GB virus-A (GBV-A) and GB virus-B (GBV-B), were recently identified in the GB hepatitis agent, and are distinct from the hepatitis A to E viruses. The putative helicase domain of GBV-A and GBV-B was found to have amino acid sequence homology with hepatitis C virus (HCV), and distantly, is also related to pestiviruses, flaviviruses, and plant viruses. A phylogenetic tree construction showed that GBVs and HCV are closely related, and they are clustered with pestiviruses, flaviviruses and plant viruses in that order.

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.

Hepatitis B virus (HBV) is classified into genotypes A-F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV.

Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus.

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.

New genotypes of TT virus (TTV) and a genotyping assay based on restriction fragment length polymorphism.

A phylogenetic analysis, using the open reading frame I sequence of 93 TT viruses (TTV) obtained from various geographical areas, indicated that the virus could be classified into six different genotypes including three hitherto unreported genotypes. The high reliability of the six clusters was confirmed by bootstrap analysis. On the basis of these sequence data, a new simple genotyping assay based on a restriction fragment length polymorphism of TTV was developed. Using the enzymes NdeI and PstI, followed by cleavage with NlaIII or MseI, it was possible to distinguish between the six TTV genotypes. This system will provide the framework for future detailed epidemiological and clinical investigations.

African origin of GB virus C/hepatitis G virus.

Ninety-four GB virus C/hepatitis G virus (GBV-C/ HGV) RNA-positive serum samples were obtained from all over the world. We found that all 15 GBV-C/HGV isolates from the Pygmies and the Bantu in the Central African region had a 12-amino acid indel (i.e. insertion or deletion) in the non-structural protein (NS) 5A region. Phylogenetic analyses of the NS5A region, using GBV-A as an outgroup, showed that these 15 isolates had diverged from the common ancestor much earlier than the remaining isolates, indicating an African origin of GBV-C/HGV.

Three different GB virus C/hepatitis G virus genotypes. Phylogenetic analysis and a genotyping assay based on restriction fragment length polymorphism.

The 5'-untranslated region (5'-UTR) sequences of 33 GB virus C/hepatitis G virus (GBV-C/HGV) obtained from different geographic areas were determined through reverse-transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5'-UTR of GBV-HGV was found to be heterogeneous, with 70.9-99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV-C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV-C/HGV isolates from Asia. Genotype-specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV-C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV-C/HGV infection.

Interferon-alpha therapy exerts selective pressure on hepatitis C virus quasispecies equilibrium.

Two patients with chronic hepatitis C virus (HCV) genotype 2b infection were studied. They responded biochemically to interferon (IFN) but had early virological and later biochemical relapse. The HCV quasispecies equilibrium in these patients was studied by a combination of cloning, sequencing and construction of phylogenetic trees. Another patient with chronic HCV genotype 2b infection was followed every 6 months for 30 months (including one episode of biochemical exacerbation) to serve as the control. Quasispecies equilibrium drifted during IFN therapy but moved back in the direction of the original equilibrium during biochemical relapse. In the control patient, there was no significant drifting throughout the follow-up period. These data suggest that IFN therapy exerts selective pressure on HCV quasispecies equilibrium.

Quantification of serum HCV core antigen by a fluorescent enzyme immunoassay in liver transplant recipients with recurrent hepatitis C--clinical and virologic implications.

BACKGROUND: Monitoring hepatitis C viremia may be useful in the management of liver transplant patients with recurrent hepatitis C virus (HCV) infection. The clinical utility of a newly described fluorescent enzyme immunoassay for the detection of serum HCV core antigen was evaluated. METHODS: Serum samples prospectively collected from 57/63 consecutive patients transplanted for HCV-related end-stage liver disease were assayed for both serum HCV core antigen by fluorescent enzyme immunoassay and HCV RNA level using a branched chain DNA signal amplification assay. HCV genotype was determined by restriction fragment length polymorphism analysis based on 5' untranslated region. One- and 2-year annual protocol liver biopsies from these patients were graded for inflammation, fibrosis, and cholestasis RESULTS: Serum HCV core antigen and HCV RNA were detected in a similar proportion of samples (256/ 281 vs. 260/281, P=NS), and there was an excellent correlation between assays (r2=0.905, P<0.0001) independent of HCV genotype. A conversion equation between HCV core antigen and HCV RNA was constructed to estimate the HCV core antigen to RNA ratio to be around 231 to 1. Mean serum HCV core antigen levels peaked initially at 3 months posttransplant but there was significant interpatient variation as to when peak levels occurred. A high serum HCV core antigen level in the first 6 months was associated with histological deterioration in terms of bridging fibrosis, cirrhosis, severe cholestasis, or retransplantation by 2-year follow-up. CONCLUSION: Determination of serum HCV core antigen level reflects HCV viremia and may have clinical implications in liver transplant patients with HCV recurrence.

Relationships between serotypes and genotypes of hepatitis B virus: genetic classification of HBV by use of surface genes.

Hepatitis B virus (HBV) surface antigen (HBsAg), which is encoded by the HBV S gene, is conventionally classified into 4 serological subtypes, adw, adr, ayw and ayr. To determine the relationship between the HBsAg seroreactivity and the nucleotide sequence diversity of the HBV S gene, the nucleotide sequences of S genes for HBV isolates reported so far were aligned with each other. The numbers of nucleotide substitutions were then estimated by the 6-parameter method, and a phylogenetic tree was constructed by the unweighted paired grouping method with arithmetic mean (UPGMA) and the neighboring-joining (NJ) method. The phylogenetic trees constructed showed that all isolates were grouped into 4 genotypes (gyw, gdw-1, gdw-2, and gdr). More importantly, the genotypes did not necessarily correspond to the conventional serotypes. In particular, serotype 'adw' can be any of genotypes gdw-1, gdw-2, or gdr. Thus, genotyping by S genes gives more accurate information about genetic variation of HBV.

Classification of hepatitis C virus into major types and subtypes based on molecular evolutionary analysis.

Molecular evolutionary analysis was applied to determine the number of hepatitis C virus (HCV) types and subtypes based on all the HCV nucleotide sequences available from the DNA data banks (DDBJ, GenBank (NCBI), EMBL) and the literature. There was an excellent concordance among the types and subtypes assigned based on different HCV genomic regions. Only one HCV isolate was assigned to different HCV types based on the 5' non-coding (NC) and envelope 1 (E1) regions. The 5' NC region was well conserved and could be used to assign only types and not subtypes. From the sequence data available there were 13 subtypes based on the core region and 14 subtypes based on the E1 and non-structural protein 5 (NS5) regions.

Genotype of GB virus C/hepatitis G virus by molecular evolutionary analysis.

GB virus C/hepatitis G virus is a newly described virus. Classification of GB virus C/hepatitis G virus into genotypes has not been established. We analyzed nucleotide sequences within the 5' untranslated region of GB virus C/hepatitis G virus isolates and segregated these isolates into genotypes. Twenty serum samples with GB virus C/hepatitis G virus RNA from Australia, Cameroon, the Congo, Japan, Mongolia, and Bangladesh were studied. Reverse transcription and polymerase chain reaction were used to obtain GB virus C/hepatitis G virus RNA. After nucleotide sequences from the 5' untranslated region were determined, 68 nucleotide sequences, including 48 previously reported sequences, were analyzed by molecular evolutionary methods. The phylogenetic tree of the 5' untranslated region showed that all strains could be divided into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3). Bootstrap analysis indicated that the strains could be divided into three major genotypes but could not be further subdivided. Moreover, frequency histograms of pairwise distances between nucleotide sequences demonstrated only one peak. These result indicated that GB virus C/hepatitis G virus can be classified into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3), and should not be divided into minor subtypes.

GB virus C/hepatitis G virus infection among Japanese patients with chronic liver diseases and blood donors.

Recently, a novel hepatitis virus, GB virus C/hepatitis G virus (GBV-C/HGV), has been isolated. To elucidate the seroprevalence of chronic GBV-C/HGV infection in Japan and the phylogenetic relationship between Japanese strains and the strains previously reported, serum GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in 203 patients with chronic liver diseases and 200 samples of voluntary blood donors. RT-PCR was performed with primers derived from the 5'-untranslated region which were conserved between GBV-C and HGV and distant from other flaviviruses including hepatitis C virus (HCV). The nucleotide sequences were determined by the dideoxy chain termination method. The phylogenetic analysis was performed by the neighbor-joining method. In 10 (4.7%) of 203 patients with chronic liver diseases and in 1 (0.5%) of 200 blood donor samples, serum GBV-C/HGV RNA was detected. Of 10 patients, 9 patients were positive for anti-HCV and negative for HBsAg, and 1 patient was positive for HBsAg and negative for anti-HCV. The phylogenetic analysis indicated that there were three major groups which were group 1 (GBV-C), group 2 (HGV), and group 3 (a group of Japanese strains). These data indicated that (1) there was a low prevalence of GBV-C/HGV infection in Japanese patients with chronic liver diseases, (2) a high proportion of patients with GBV-C/HGV infection had chronic HCV infection however, and (3) there were at least three groups in strains of GBV-C/HGV.

High prevalence of GB virus C/hepatitis G virus infection among the Jewish population in Uzbekistan.

Although a new virus, GB virus C/hepatitis G virus (GBV-C/HGV), has been isolated from patients with hepatitis by two different research groups, its prevalence in the world and pathogenesis are still unknown. In this study, 92 samples from the Jewish population of Uzbekistan were investigated for the prevalence of GBV-C/HGV. GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR) using specific primers derived from the 5'-untranslated region (5'-UTR). Sequences were analyzed by a molecular evolutionary method. Of 92 samples, GBV-C/HGV RNA was detected in ten (10.9%), HCV RNA was present in two (2.2%), and HBsAg in eight (8.7%). HTLV-I and HIV infection was not detected. Single GBV-C/HGV infection was detected in eight (80%), and co-infection with HBV or HCV was detected in only two of the GBV-C/HGV infections. Alanine aminotransferase (ALT) levels were elevated in three (3.3%), but none with single GBV-C/HGV infection had an elevated ALT level. Nine people (90%) with GBV-C/HGV infection were distributed under the mean age of the population (P < 0.05). Molecular evolutionary analysis showed all GBV-C/HGV strains in this study were related to the HGV derived from the US. These results indicate that (1) GBV-C/HGV infection is highly prevalent among the Jewish population in Uzbekistan; (2) single GBV-C/HGV infections without persistent hepatitis are common; and (3) GBV-C/HGV infection is present among the younger generation.

GB virus C/hepatitis G virus infection among Colombian native Indians.

To elucidate the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection in Colombian native Indians, serum GBV-C/HGV RNA was assayed in 163 native Indians and 67 members of the general population in Colombia. The native Indians (males:females = 40:123) and the members of the general population (males:females = 20:47) were tested by reverse transcription-semi-nested polymerase chain reaction. Of the 163 native Indians, 10 (6.1%) were positive for GBV-C/HGV RNA, compared with one (1.5%) of 67 from the general population. All Indians were negative for hepatitis B surface antigen and antibody to hepatitis C virus. Of 10 Indians with GBV-C/HGV RNA, the genotype of nine subjects was the Asian type. These data indicated that 1) the prevalence of GBV-C/HGV RNA in Colombian native Indians is high, and 2) GBV-C/HGV was probably brought from Asia and inherited for generations in some native Indian groups.

Serologically defined genotypes of hepatitis C virus among Japanese patients with chronic hepatitis C.

Recently, Tsukiyama-Kohara et al. (1993) reported that most hepatitis C viruses (HCVs) in Japan can be classified into two types, type 1 and type 2, on the basis of the NS4 region nucleotide sequence. They developed a new assay in which antibodies against group-specific recombinant proteins of the NS4 region were measured by ELISA (serologically defined genotype, serotype). In the present study, we examined 306 patients with chronic liver disease due to HCV infection. The sensitivity of this assay was 98.7% (302/306). The serotype distribution of HCV was 230/306 (75.2%) for type 1, 65/306 (21.2%) for type 2, 7/306 (2.3%) for mixed, both being positive, and 4/306 (1.3%) indeterminate. The frequency of type 1 was significantly higher than that of type 2 (P < 0.01). There were no significant differences in clinical characteristics among the mixed and the indeterminate serotypes. Among the mixed-serotype patients, 4/7 (57.1%) showed seroconversion to a single serotype at 6 and 9 months later, although the serotypes of the indeterminate-serotype patients were also indeterminate at 6 and 9 months later. Using aliquots of the same serum samples, HCV genotyping was carried out by the reverse transcription polymerase chain reaction (RT-PCR) method using type-specific primers derived from the NS5 region of HCV to verify the specificity of this serotyping. The sensitivity of genotyping by RT-PCR was 167/183 (91.3%). The HCV genotypes determined by both methods were consistent in 161/183 (88.0%) of the cases, and there were no contradictory results for any sample between the two methods. These findings indicate that serological genotyping might be useful in determining the HCV genotype among Japanese patients with HCV infection.

Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers.

Hepatitis B virus (HBV) has been classified into six genotypes designated A-F by sequence divergence in the entire genome exceeding 8%. Very recently, the seventh genotype was reported and named genotype G. HBV genotype G is distinct from genomes of the other six genotypes in that it possesses an insertion of 36 nucleotides in the core gene, and has been found so far in France and the United States. A method for determining HBV genotype G was developed by polymerase chain reaction (PCR) with primers deduced from the 36-nucleotide (nt) insertion in five isolates of HBV genotype G the sequences of which have been deposited in DNA databases. The validity of this method, for specifically detecting HBV genotype G, was verified on a panel consisting of 142 HBV isolates of six major genotypes and four of genotype G. A total of 540 sera containing HBV in Japan covering symptom free carriers and patients with a spectrum of chronic liver disease were tested by this method, but not a single HBV genotype G sample was found. A possible method for serological determination of hepatitis B surface antigen of genotype G is suggested, without amplification or sequencing nucleotides, which would expand epidemiological and clinical researches on HBV genotype G.