Erwin Sablon

Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes

International standardization and coordination of the nomenclature of variants of hepatitis C virus (HCV) is increasingly needed as more is discovered about the scale of HCV‐related liver disease and important biological and antigenic differences that exist between variants. A group of scientists expert in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and Los Alamos (United States), met to re‐examine the status of HCV genotype nomenclature, resolve conflicting genotype or subtype names among described variants of HCV, and draw up revised criteria for the assignment of new genotypes as they are discovered in the future. A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name reassignments to create consistency in nomenclature. The paper also contains consensus proposals for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. A proposal was made that HCV variants be classified into 6 genotypes (representing the 6 genetic groups defined by phylogenetic analysis). Subtype name assignment will be either confirmed or provisional, depending on the availability of complete or partial nucleotide sequence data, or remain unassigned where fewer than 3 examples of a new subtype have been described. In conclusion, these proposals provide the framework by which the HCV databases store and provide access to data on HCV, which will internationally coordinate the assignment of new genotypes and subtypes in the future. (HEPATOLOGY 2005.)

HBsAg seroclearance in chronic hepatitis B in the Chinese: Virological, histological, and clinical aspects

Few studies have examined Chinese patients with chronic hepatitis B who exhibit hepatitis B surface antigen (HBsAg) seroclearance. We comprehensively studied the biochemical, virological, histological, and clinical aspects of 92 patients with HBsAg seroclearance (median follow‐up, 126 months). Ninety‐two HBsAg‐positive controls matched for age, sex, and duration of follow‐up were also recruited. Liver biochemistry, serum hepatitis B virus (HBV) DNA levels, and development of clinical complications were monitored. Intrahepatic total and covalently closed circular (ccc) HBV DNA were measured quantitatively in 16 patients. HBV genotype was determined in 30 patients. The mean age at HBsAg seroclearance was 48.8 (+ 13.81) years. There was a significant improvement in serum alanine aminotransferase levels after HBsAg seroclearance (p<0.0001). Patients with genotype B had a higher chance of HBsAg seroclearance than those with genotype C (P = .014). Ninety‐eight percent of patients had undetectable serum HBV DNA. Thirty‐seven percent of patients had low titer of intrahepatic HBV DNA, mainly in the form of cccDNA (71%‐100%). All 14 patients with liver biopsies had near normal histology. There was no difference in the risk of development of hepatocellular carcinoma (HCC) between patients with and without HBsAg seroclearance. However, the mean age of HBsAg seroclearance was significantly older in patients with HCC than in patients without HCC (P = .016). In conclusion, patients with HBsAg seroclearance had favorable biochemical, virological, and histological parameters. Intrahepatic HBV DNA level was low and predominantly in the form of cccDNA. However, HCC could still develop, particularly in patients with cirrhosis who had HBsAg seroclearance at an older age. (HEPATOLOGY 2004;39:1694–1701.)

Monitoring Drug Resistance in Chronic Hepatitis B Virus (HBV)-Infected Patients during Lamivudine Therapy: Evaluation of Performance of INNO-LiPA HBV DR Assay

ABSTRACT Sensitive and early detection of emerging hepatitis B virus (HBV) drug resistance may not only help monitor the viral dynamics associated with lamivudine treatment but could also improve therapeutic decision making. This is especially important when new antivirals effective against lamivudine-resistant HBV become available. A total of 159 serum samples from 33 chronic HBV patients receiving lamivudine treatment were analyzed at four centers for the presence of lamivudine-resistant mutations at codons 528 [180] (proposed revised nomenclature according to Stuyver et al. [Hepatology 33:751-757, 2001] shown in brackets), 552 [204], and 555 [207] of the HBV polymerase. Sequencing data were compared with results generated by the INNO-LiPA HBV DR line probe assay (LiPA), an assay based on reverse hybridization of amplified HBV DNA fragments with specific nucleotide probes immobilized on nitrocellulose strips. LiPA provided at least the same information as sequencing for 97.5% of all codons analyzed for codon 528 [180], 95% for codon 552 [204], and 100% for codon 555 [207]. The most common reason for discrepant or indeterminate results (0.4% and 1.5%, respectively) in a small percentage of the population tested could be attributed to polymorphisms not yet covered by LiPA probes. In at least five patients, a mutant could be detected earlier by LiPA than by sequencing. In 15 patients, LiPA detected mixed wild-type and mutant virus populations before viral breakthrough. These results demonstrate that INNO-LiPA HBV DR is a highly sensitive and easily applicable assay for the detection and monitoring of lamivudine-resistant mutations in chronic hepatitis B patients and that the assay is more sensitive than sequencing in detecting mixed mutant and wild-type sequences.

A comprehensive system for consistent numbering of HCV sequences, proteins and epitopes

InOctober 2004, an expert meeting was convened in parallel with the 11th Symposium onHepatitis C and Related Viruses to discuss how HCV sequence databases could introduce and facilitate a standardized numbering system for HCV nucleotides, proteins and epitopes. Inconsistent and inaccurate numbering of locations in DNA and protein sequences is a problem in the HCV scientific literature. Consistency in numbering is increasingly required for functional and clinical studies of HCV. For example, an unambiguous method for referring to amino acid substitutions at specific positions in NS3 and NS5B coding sequences associated with resistance to specific HCV inhibitors is essential in the investigation of antiviral treatment. This article provides a practical guide to help circumvent these problems in the future, and to bring a common language into discussions in the field. The scope of the current system is limited to the HCV polyprotein and the untranslated regions (UTRs); because of the controversial nature and extreme length variation of the alternate reading frame proteins, numbering for these proteins, if needed, will be decided at a later date. We propose a numbering system adapted from the Los Alamos HIV database,1 with elements from the hepatitis B virus numbering system.2 The system comprises both nucleotides and amino acid sequences and epitopes. It uses the full length genome sequence of isolate H77 (accession number AF009606) as a reference, and includes a method for numbering insertions and deletions relative to this reference sequence. H77 was chosen because it is a commonly used reference strain for many different kinds of functional studies. Furthermore, RNA transcripts from this sequence are of demonstrated infectivity,3,4 providing evidence that the 5 and 3 ends of the sequence are complete. Table 1 lists the boundaries of HCV genomic regions and Fig. 1 provides detailed nucleic acid and amino acid numbering over the complete AF009606 HCV genome sequence.

Rapid and Sensitive Assays for Determination of Hepatitis B Virus (HBV) Genotypes and Detection of HBV Precore and Core Promoter Variants

ABSTRACT Hepatitis B virus (HBV) genotypes may influence HBeAg seroconversion rates, mutational patterns in the precore (PC) and core promoter (CP) regions, severity of liver disease, and response to antiviral treatment. Development of rapid, simple, and standardized assays to detect viral genotypes and common mutations in the PC and CP regions can accelerate research on the clinical significance of these variants. We aim to assess the accuracy of a line probe assay in determining HBV genotypes and detecting HBV PC and CP variants. HBV genotypes in 701 patients and PC and CP variants in 600 patients with chronic HBV infection from China and the United States were studied using the INNO-LiPA assay. All but one (99.9%) sample were classified by the genotyping assay. All eight genotypes, i.e., A to H, were found. The INNO-LiPA genotyping assay results were completely concordant with those of sequencing. Using the INNO-LiPA PC assay, 99.8 and 94.7% samples were classifiable in the PC and CP regions, respectively. The PC assay results were completely concordant with those of sequencing in all samples that showed either wild-type or variant sequence. The line probe assay was more sensitive in detecting mixtures than was direct sequencing. By INNO-LiPA, only 50 and 27% of the samples, with mixed wild-type and variant sequence in the PC and CP region, respectively, showed mixed sequence by direct sequencing. INNO-LiPA is rapid, sensitive, and reliable—thus enabling accurate determination of HBV genotypes and detection of PC and CP variants in a large population of patients.

Evaluation of Versant Hepatitis C Virus Genotype Assay (LiPA) 2.0

ABSTRACT Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5′ untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturers instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison study, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.

The relationship between HBV‐DNA levels and cirrhosis‐related complications in Chinese with chronic hepatitis B

Summary.  We studied the hepatitis B virus (HBV)‐DNA levels below which the development of cirrhosis‐related complications became unlikely in chronic hepatitis B (CHB). Seventy‐nine Chinese CHB patients with cirrhosis‐related complications and 158 age‐, sex‐ and HBeAg status‐matched patients without complications were enrolled. The precore and core promoter mutations were detected by the Line Probe assay (LiPA). HBVDNA levels were determined by Digene assay and Cobas Amplicor Monitor test. Patients with complications had higher HBVDNA levels than those without complications (P = 0.02). HBeAg‐positive patients with complications had similar alanine transferase (ALT) and HBVDNA levels and frequency of precore mutations, but higher frequency of core promoter mutations (P = 0.003), compared with those without complications. Anti‐HBe‐positive patients with complications had higher ALT and HBVDNA levels (P < 0.01) but similar frequency of precore and core promoter mutations, compared with those without complications. Anti‐HBe patients (24.5%) with complications had HBVDNA levels <104 copies/mL. The major factor for the development of cirrhotic complications was viral loads but cirrhotic complications continued to develop in patients with HBVDNA levels below 104 copies/mL.

Relationship between the Development of Precore and Core Promoter Mutations and Hepatitis B e Antigen Seroconversion in Patients with Chronic Hepatitis B Virus

Chinese patients with chronic hepatitis B virus (332 with and 44 without cirrhosis-related complications) were studied. Fifty percent of patients <30 years old had precore mutations. The prevalence of precore mutations among hepatitis B e antigen (HBeAg)-positive patients, although lower than that among anti-HBe-positive patients (P=.031), was already high (44.2%). Median HBV DNA level in anti-HBe-positive patients was 1.5 x 10(6)-1.55 x 10(6) copies/mL, irrespective of the presence or absence of precore mutations. There was no difference in the prevalence of precore mutations between patients with and without complications (P, not significant). However the prevalence of core promoter mutations was higher among patients with complications than among those without complications (90.5% vs. 69.3%, respectively; P=.003). In conclusion, precore mutations occurred in a large proportion of Chinese patients with chronic hepatitis B virus before HBeAg seroconversion. The development of complications was not related to precore mutations but was probably due to the persistence of significant viremia after HBeAg seroconversion.

Sensitive line probe assay that simultaneously detects mutations conveying resistance to lamivudine and adefovir

ABSTRACT The INNO-LiPA HBV DR v2 assay is designed to detect hepatitis B virus mutations conveying resistance to lamivudine and adefovir. Our study confirms that this assay can simultaneously detect the presence of lamivudine and adefovir resistance mutations in clinical samples, has a high degree of concordance with sequencing, and can detect mutants earlier.

Interferon and Ribavirin Therapy for Chronic Hepatitis C Virus Genotype 6: A Comparison with Genotype 1

Because there is a lack of data on the treatment outcome of patients who carry hepatitis C virus (HCV) genotype 6, we conducted a prospective study, to compare the effect of interferon and ribavirin therapy in HCV genotypes 1 and 6, of patients with seropositive anti-HCV, persistently elevated alanine transaminase levels, and detectable HCV RNA. Patients were treated with subcutaneous recombinant interferon alpha-2b and ribavirin for 12 months. Of 40 patients, 16 had genotype 6, and 24 had genotype 1. An end-of-treatment response was detected in 12 (75%) patients with genotype 6 and in 10 (41.6%) patients with genotype 1 (P=.05). A sustained virological response (SVR) was present in 10 (62.5%) patients with genotype 6 and in 7 (29.2%) patients with genotype 1 (P=.04). Genotype 6 has a better response than genotype 1 and is associated with a higher SVR.