Koichi Kanai

Hepatitis C virus infection and mutations of mannose-binding lectin gene MBL

SummaryWe assessed the genetic polymorphism of mannose-binding lectin (MBL) in 93 patients with chronic hepatitis C (45 responders and 48 nonresponders to interferon) and 218 healthy controls. Mutant allele was identified only at codon 54 (Gly→Asp), leading to three genotypes (54 m/m, 54 W/m, and 54 W/W). Frequency of 54 m/m was significantly lower in interferon-responders (2.2%) compared to those in nonresponders (14.6%) and controls (10.6%): p<0.05. Our results suggest that homozygous carriage of the variant allele of codon 54 of MBL may predict poor response to interferon in chronic hepatitis C patients.

Clinical implications of mutations C-to-T1653 and T-to-C/A/G1753 of hepatitis B virus genotype C genome in chronic liver disease.

SummaryAmong many mutational “hot spots” on hepatitis B virus (HBV) genome, A-to-T1762 and G-to-A1764 within the core promoter have been underscored in view of disease association as well as viral expression/replication. Although to a lesser extent, C-to-T1653 and T-to-V(C/A/G)1753 were also noteworthy in our previous study. To assess the clinical significance of these mutations, we determined the nucleotide sequence of an HBV DNA fragment covering these sites in HBsAg-positive blood donors (n=160) and patients with chronic hepatitis (n=66), liver cirrhosis (n=45), and hepatocellular carcinoma (n=58), most of whom were infected with genotype C HBV (subtype adr). In cases where HBe antigen was positive, the frequency of T1653 and/or V1753 showed a striking increment from chronic hepatitis patients (18%) to liver cirrhosis and/or hep- atoma patients (82%), whereas that of T1762/A1764 was already high in chronic hepatitis patients (76%). In HBe antigen-negative cases, by contrast, significant difference in the frequency of T1653/V1753 mutants was found between blood donors (22%) and chronic hepatitis patients (67%). Our results suggest that T1653/V(particularly C)1753 mutants are more closely associated than T1762/A1764 with the progression of liver disease from chronic hepatitis to cirrhosis in HBe antigen-positive patients. A system of site-directed mutagenesis PCR RFLP was constructed to diagnose T1653 and C/A1753 more conveniently. Detecting T1653 and C/A1753 by this method would contribute to the differential diagnosis of HBV-associated liver disease.

Genotypes of TT virus (TTV) compared between liver disease patients and healthy individuals using a new PCR system capable of differentiating 1a and 1b types from others

Summary TT virus (TTV) lacks obvious pathogenicity; almost all of the infected hosts are symptom-free. A possibility remains, however, that certain strains can cause liver disease while most others are non-pathogenic. Genotypes 1 a and 1 b have been proposed to contain such pathogenic strains. To test this possibility, we constructed a PCR system capable of detecting TTV of the 1 a and 1 b genotypes differentially from the other genotypes, and compared the frequencies of these genotypes between patients with liver disease of unknown etiology (n=42) and healthy individuals (n=50). The assay comprised 3 steps: i) PCR to amplify a 3.2-kb fragment using universal primers; ii) 2nd-round PCR, starting from the 3.2-kb amplicon, for a ∼280-nt fragment by use of genotype 1-specific primers; and iii) digestion of the ∼280-nt amplicon with MboI and BanI to discriminate between 1 a and 1 b. Eventually, 40 (95%) of the patients and 47 (94%) of the controls were positive for the 3.2-kb amplicon, and the 1 a, 1 b, 1 a+1 b, and non-1 genotypes of TTV were found in 2 (5%) vs 4 (9 %), 5 (13%) vs 4 (9%), 1 (3%) vs 1 (2%) and 32 (80%) vs 38 (81%) of the 40 patients and 47 controls, respectively: the distribution was almost identical between the two groups. The hypothesis that the genotype 1 of TTV may be more closely associated with liver disease than other genotypes was not supported by this study.

Improvement of proteinuria in a case of hepatitis B-associated glomerulonephritis after treatment with interferon

Disappearance of proteinuria was observed in a patient with hepatitis B-associated chronic glomerulonephritis after treatment with leukocyte interferon. The decrease of proteinuria was preceded by the disappearance of both deoxyribonucleic acid polymerase activity and hepatitis B e antigen from the patients sera.

Distribution of the hepatitis B surface antigen subtypes in indonesia : implications for ethnic heterogeneity and infection control measures

SummaryPrevious studies on the distribution of subtypes of hepatitis B surface antigen (HBsAg) in Indonesia have not entirely covered the whole nation. Consequently, we determined the HBsAg subtypes, adw, adr, ayw, and ayr in a total of 569 HBsAg-positive sera from areas so far not studied. With results in this and our previous studies taken together (a total of 3045 HBsAg-positive sera were analyzed), a nationwide picture on the HBsAg subtype distribution indicated that Indonesia could roughly be divided into 4 zones: (i) adw-predominant zone consisting of Sumatera, Java, southern part of Kalimantan, Bali, Lombok, Ternate, and Morotai; (ii) ayw-zone of the eastern part of Nusa Tenggara and Moluccas, (iii) adr-zone of Irian Jaya; and (iv) a mixed subtype-zone of Kalimantan, Sulawesi and Sumbawa. An interesting exception was Padang of Sumatera island: Padang was rich in adr although it is far from the adr-zone. Since the HBsAg subtype, like mutations in mitochondrial DNA, could be used as an ethnological marker, our present results suggest that the peoples in Indonesia have at least three major ethnic origins, represented by adw, ayw, and adr. Of another note, the diagnostic and preventive measures for hepatitis B virus (HBV) infection in Indonesia may be improved by considering such diversity of HBV strains revealed in this study.

Interferon-α2a for chronic hepatitis B with e antigen or antibody: comparable antiviral effects on wild-type virus and precore mutant

Summary. Recombinant interferon‐α2a (IFN‐α2a) in a total dose of 702 MU was given to 31 patients: nine with wild‐type hepatitis B virus (HBV) and hepatitis B e antigen (HBeAg) (A); four with HBeAg and a mixed infection with wild‐type HBV and precore mutants (B); 11 with antibody to HBeAg (HBeAb) and a mixed infection (C); and seven with HBeAb and precore mutants alone (D). HBV DNA was not cleared in any patient in groups A and B. By contrast, in patients with HBeAb, HBV DNA was ultimately lost in four patients in group C, as well as in 10 patients in group D. Thus, patients with HBeAb and infected with precore mutants alone (D) lost serum HBV DNA more often than those with HBeAg and wild‐type HBV (A). Patients with low pre‐treatment levels of HBV DNA cleared virus more frequently, and the response of precore mutant to IFN was comparable with that of wild‐type HBV in patients who had a mixed infection. Based on these results, precore mutants do respond to IFN, and therefore, IFN is indicated in patients with HBeAb, especially those with low serum HBV DNA levels.

Muscle cramps in chronic liver diseases and treatment with antispastic agent (eperisone hydrochloride)

To The Editor: Muscle cramps, which can be defined as involuntary and painful contractions of skeletal muscles, frequently occur in a variety of pathologic conditions related to neuromuscular, endocrine, or metabolic disease (1, 2), although muscle cramps sometimes occur in healthy persons. Among patients with liver disease, muscle cramps are most frequently seen in individuals with cirrhosis. Konikoff and Theodor reported that 88% of cirrhotic patients had experienced muscle cramps (3). We have prospectively determined the incidence of muscle cramps in patients with chronic liver diseases and in healthy individuals and have evaluated the effectiveness of treatment with eperisone hydrochloride, an antispastic agent (4), on muscle cramps in cirrhotic patients. One hundred seventy-six consecutive patients with chronic liver disease (103 males, 73 females, median age 54 years) visiting the liver clinic of Hamamatsu University Hospital and 142 healthy subjects (63 males, 79 females, median age 54 years) were examined. The group with chronic liver disease consisted of 80 patients with cirrhosis (44 males, 36 females, median age 59 years) and 96 patients with chronic hepatitis without cirrhosis (59 males, 37 females, median age 50 years). Patients with alcoholic liver injury were excluded because of the possibility that muscle cramps in alcoholics could be associated with alcoholic myopathy and/or neuropathy. No patients had drug-induced liver injury or any malignant neoplasm. Each subject was questioned about the frequency and localization of muscle cramps using a standardized questionnaire. Serum electrolytes (Na, K, CI, Ca) were measured in all of the cirrhotic patients, since a substantial number of these patients were taking diuretics flurosemide and/or spironolactone) for ascites and peripheral edema. An oral glucose tolerance test (75 g load) was also performed in all cirrhotic patients except for those who were known to be diabetic. The incidence of muscle cramps in patients with chronic liver diseases and healthy subjects is shown in Table 1. Twenty-five of 80 cirrhotics (31%) cornplained of frequent muscle cramps (more than once a week), while only 10 of 142 healthy subjects (7%) reported such a high frequency. The incidence of muscle cramps in the patients with chronic hepatitis without cirrhosis was 5%, which was not statistically different from healthy subjects. Most of muscle cramps (63% in the cirrhotics, 72% in patients with chronic hepatitis without cirrhosis, and 81% in healthy subjects) occurred in the calves. The incidence of muscle cramps in the toes, fingers, and thighs were 23%, 25%, and 19% for the cirrhotics, compared to 32%, I I%, and 5% for healthy subjects. The cirrhotic patients had a higher incidence of muscle cramps in the fingers or thighs than the patients with chronic hepatitis without cirrhosis or healthy subjects (P < 0.01). Serum electrolyte (Na, K, CI, Ca) concentrations as well as the administration of diuretics did not correlate with the frequency of muscle cramps in the patients with liver cirrhosis. There was no significant difference in the incidence of muscle cramps between cirrhotic patients with or without diabetes (27% versus 23%, respectively). To determine the efficacy of eperisone hydrochloride in the treatment of muscle cramps, doses of 150-300 mg per day were given orally to 21 of the 25 cirrhotic patients reporting frequent muscle cramps (more than once a week). This research was carried out in accordance with the Helsinki Declaration. Three of the remaining patients refused treatment, and one patient was not given the drug due to renal dysfunction. The frequency of muscle cramps was evaluated before and after eight weeks of treatment. Of 18 patients who continued the treatment for eight weeks, muscle cramps completely disappeared in 11 patients (61%), decreased in frequency in six (33%), and were unaltered in only one patient. Side effects were observed in four patients: fatigue occurred in one, epigastric discomfort in two, and dizziness in one patient. In three of these patients, the treatment was discontinued. No change in liver function tests was detected. The results of our study confirm the previous report that patients with liver cirrhosis experience a higher frequency of muscle cramps than healthy subjects (3). Although the pathophysiology of muscle cramps in liver cirrhosis still remains obscure, a suitable treatment for these distressing symptoms is

Antithrombin III concentrate in the treatment of fulminant hepatic failure.

SummaryTwenty-six patients with fulminant hepatic failure were treated with daily infusions of antithrombin III concentrate until recovery of consciousness or death. Seven patients were alive (group A), 7 survived 17 to 47 days after treatment (group B), and 12 died within 9 days (group C). Decreased plasma antithrombin III levels increased on the day after treatment, irrespective of the pretreatment levels in all patients. Continuous or temporary normalization was seen in all patients in groups A and B, but in only 5 in group C patients whose bleeding was extensive (p<0.05). An abrupt drop in peripheral platelet counts occurred when plasma antithrombin III levels were below normal. General bleeding accompanied this drop. These results suggest that maintained normal plasma antithrombin III levels are beneficial for prolonged survival time in fulminant hepatic failure, probably through controlling intravascular coagulation, and that antithrombin III infusion may be useful for such treatment.

Response to Interferon-α2a in Patients with e Antigen-negative Chronic Hepatitis B

Sixty-eight consecutive patients with chronic hepatitis B received 702 million units of recombinant interferon-alpha 2a. Of the 24 patients negative for hepatitis B e antigen (HBeAg) in serum, the normalization of serum transaminase occurred in 14 (58%) at the completion of interferon therapy and in 13 (54%) at 12 months thereafter; it was normalized in 17 (39%) and 13 (30%), respectively, of the 44 HBeAg-positive patients. Of the HBeAg-negative patients, hepatitis B virus DNA was cleared from serum in six (25%) at the completion and in one (4%) at 12 months thereafter, in contrast to only one (2%, p < 0.05) and none of the HBeAg-positive patients, respectively. The 1896th nucleotide of G (G1896) for codon 28 for tryptophan or A (A1896) for the stop codon 28 in the precore region was determined by restriction fragment length polymorphism. The ten HBeAg-negative patients with A1896 only in the precore region had lower pretreatment levels of viral markers, which decreased more rapidly and extensively after interferon than in the 14 HBeAg-negative patients with a mixture of G1896 and A1896 or in the 44 HBeAg-positive patients. These results indicate that patients with HBeAg-negative chronic hepatitis B may respond better to interferon than HBeAg-positive patients, and that the precore mutant with the stop codon 28 may be sensitive to interferon.

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

SummaryEfficacy 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 IFNα2a 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 significant difference (32% vs 56%, p=0.034). SR rate in group III was 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 IFNα 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.