Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.     

Hepatitis B surface antigen (HBsAg) subtype-specific antibodies in persons vaccinated against hepatitis B

One hundred sixty-three persons immunised against hepatitis B with a vaccine containing HBsAg either of adw or ayw subtype were examined for antibodies against the a, d, and y determinants of HBsAg. Sera were tested for antibodies against HBsAg adw and HBsAg ayw separately by a solid-phase radioimmunoassay using polystyrene beads coated with HBsAg of either adw or ayw subtype, and the relative amounts of antibodies against the single determinants were calculated. After the third immunisation, all vaccinees had antibodies against the common determinant a. A quantitative evaluation showed that on average about 50% of HBsAg-specific antibodies were directed against the a determinant, and about 50% against d or y, respectively. However, as only anti-a is protective against cross-infection with other HBsAg subtypes, the degree of immunity of a person vaccinated against hepatitis B should be evaluated by the determination of antibodies to a rather than antibodies against total HBsAg.     

Hepatitis B virus genotypes and HBsAg subtypes in refugees and injection drug users in the United States determined by LiPA and monoclonal EIA.

Hepatitis B virus (HBV) genotyping and hepatitis B surface antigen (HBsAg) subtyping were carried out on sera from 196 HBsAg-positive patients, including 151 refugees entering the United States and 45 injection drug users in Seattle. HBsAg subtyping was performed by enzyme immunoassay (EIA) using a panel of monoclonal antibodies and the HBV genotype was determined by polymerase chain reaction (PCR) followed by detection of amplified HBV DNA by a reverse-phase hybridization line probe assay (LiPA) using genotype-specific probes. HBV DNA was detected by PCR in 155 (79%) of the 196 sera and all 155 were genotyped by LiPA. Samples from Southeast Asia were predominantly genotype B/subtype ayw1 and genotype C/adr; samples from the former Soviet Union and eastern Europe were mostly genotype D/ayw2 and genotype D/ayw3; samples from east Africa were mainly genotype A/adw2 and genotype D/ayw2; and samples from injection drug users were mostly genotype D/ayw3 and genotype A/adw2. Some strains of ayw3 gave atypical monoclonal antibody reactivity patterns in the subtyping assay due to a Val/Ala instead of a Thr at amino acid residue 118 and a Thr instead of a Met at residue 125. A strain of ayw2 also gave an atypical monoclonal antibody reactivity pattern due to an Ala instead of a Thr at amino acid residue 123. LiPA genotyping and monoclonal EIA subtyping can provide useful information for epidemiological studies.     

Comparison of the technical and clinical performance of the Elecsys® HBsAg II assay with the Architect®, AxSym®, and Advia® Centaur HBsAg screening assays

South East Asia has some of the highest prevalence rates of hepatitis B virus (HBV) infection (≥8%) in the world, and the emergence of hepatitis B surface antigen (HBsAg) mutant strains is a growing problem. Assays with the highest levels of sensitivity, including mutant detection, should be used for routine HBsAg screening. In this large multicenter study, the clinical and technical performance of the fully automated Elecsys HBsAg II assay was compared with the Architect, AxSYM, and Advia Centaur HBsAg assays for HBsAg screening. Nine laboratories (three each from Thailand, Korea, and Singapore) compared the Elecsys HBsAg II assay with their routine HBsAg screening assay against a range of stored and routine clinical samples, including recombinant mutants. The Elecsys HBsAg II assay demonstrated equivalent sensitivity and specificity to the Architect HBsAg assay. However, the Elecsys HBsAg II assay recognized a native mutant sample (L94S, L97V, L98V, T123A) that the Architect HBsAg assay failed to detect. The AxSYM and Advia Centaur HBsAg assays appeared less sensitive for the detection of early HBV infection and also failed to detect some of the recombinant mutant strains. There was almost complete agreement between the Elecsys HBsAg II assay and comparator assays with respect to routine serum samples. The results of this study demonstrate that the Elecsys HBsAg II assay is a highly sensitive and specific screening assay for HBsAg and detects reliably the most important and clinically relevant HBV mutants and genotypes. It is suitable for routine HBsAg screening in Asia. J. Med. Virol. 82: 755–762, 2010. © 2010 Wiley‐Liss, Inc.     

Improved ELISA for the detection of HBsAg

A sandwich ELISA for hepatitis B virus surface antigen (HBsAg) was developed using monoclonal anti-HBs for the solid phase and horse-radish peroxidase labelled sheep anti-HBs.

The sensitivity was approx. 0.3 U/ml HBsAg, in the standard test procedure,comprising two incubation steps of 1 h at 37°C, or in a shortened procedure comprising two incubation steps of 30 min at 50°C. A slightly reduced sensitivity (approx. 0.5 U/ml) was obtained when the two incubations were combined in a one-step incubation for 1 h at 37°C. All three procedures were completed by an incubation for 30 min at room temperature with peroxide and tetra-methylbenzidine.

The number of false positives obtained when donor sera were screened was below 0.5%. False positive reactions occurred more frequently, but still to a limited extent, when previously selected sera containing rheumatoid factor or other possibly interfering factors were tested with the standard procedure. Most sera containing factors that interfere with a commercial ELISA for HBsAg using sheep anti-HBs coated plates, were negative. Rheumatoid factor positive sera seldom gave false positive results.

The lower detection limit became approx. 0.1 U/ml when the cut-off was reduced, while the number of false positives in a donor population only increased to 1.5%. The results obtained with reagents from four different batches indicate a stability of up to 4 wk at 37°C and for at least 26 wk at 4°C.     

Non-A,non-B hepatitis in Palermo,Western Sicily

Aetiological studies were carried out on 407 cases of acute viral hepatitis during two consecutive years in two general hospitals in Palermo, Sicily. Two hundred ninety-seven showed serological evidence of hepatitis A virus (HAV) infection and 73 of hepatitis B virus (HBV) infection. Of the remaining 37, two had a serologically diagnosed cytomegalovirus hepatitis, while 35 were classified as non-A, non-B (NANB) hepatitis. The frequency of the different major agents of acute viral hepatitis was notably influenced by the age of the patients, HAV being prevalent in children and HBV and NANB in adults. About one-fourth of the adult cases of sporadic, acute viral hepatitis were attributed to NANB virus(es). The acute disease appeared less severe than B hepatitis, as indicated by the duration of jaundice, peak serum bilirubin, and aminotransferase levels. No history of drug addiction or of parenteral exposure to blood or blood products was obtained from the presumed NANB hepatitis patients.     

e抗原阳性慢性乙型肝炎患者血清HBV-DNA与HBsAg、HBeAg的相关性分析

目的 研究e抗原阳性慢性乙型肝炎患者外周血中HBV-DNA载量与乙型肝炎病毒表面抗原（HBsAg）、乙型肝炎病毒e抗原（HBeAg）的相关性,及其在不同性别、年龄群体中的差异.方法 收集319例e抗原阳性慢性乙肝患者血清,采用实时荧光定量PCR法检测HBV-DNA载量,用时间分辨免疫荧光法检测HBsAg和HBeAg的浓度,利用SPSS软件做统计分析.结果 HBV-DNA载量与HBsAg含量有良好的相关性（r=0.514,P〈0.001）;与HBeAg含量有相关（r=0.337,P〈0.001）;女性的HBeAg水平要高于男性患者（P〈0.05）;年龄（31～50）岁组、〉50岁组的HBV-DNA、HBsAg 及HBeAg值皆高于年龄 〈30岁组 （P〈0.001）.结论 e抗原阳性慢性乙型肝炎患者血清中HBV-DNA载量与HBsAg、HBeAg定量水平皆有相关性,其中与HBsAg相关性更佳.     

HBV-LP与HBV-DNA联检的临床应用

目的：探讨血清乙型肝炎病毒大蛋白与HBV-DNA联检在乙型肝炎患者诊治中的意义。方法：对162例HBV感染者及47名健康对照组血清采用ELISA检测乙型肝炎病毒大蛋白及乙型肝炎病毒标志物;FQ-PCR定量检测HBV-DNA。结果：162例HBV感染者血清中,HBV-LP浓度与HBV-DNA拷贝数间具有良好的正相关性（rs=0.64,P〈0.001）,不同HBV-DNA拷贝数组别间HBV-LP浓度存在差异显著性（P〈0.01）;HBV-LP与HBV-DNA、HBeAg间均关联显著（P〈0.01）。HBV-LP与HBV-DNA、HBeAg间阳性率均存在差异显著性（P〈0.05）,HBV-LP阳性率为84.57%,较HBV-DNA、HBeAg均敏感。其中HBV-LP在HBV-DNA阳性组中阳性率为91.18%（93/102）,DNA阴性组中阳性率为73.33%,均较HBeAg高;在58例HBV-DNA和HBeAg共同阴性的HBV感染血清中,HBV-LP检出42例阳性。结论：血清HBV-LP浓度与HBV-DNA联检有利于提高HBV感染者体内病毒复制、疾病进程、疗效与预后判断的血清学诊断与监测水平。     

Kinetics of HBV DNA and HBsAg in acute hepatitis B patients with and without coinfection by other hepatitis viruses

The kinetics of hepatitis B virus (HBV) and its surface antigen (HBsAg) during acute hepatitis has not yet been studied accurately in a representative number of patients. The influence of coinfecting hepatitis viruses during the acute phase of infection is not known. Three to four serum samples from 21 patients with acute HBV monoinfection and 27 with coinfection were taken at intervals of 6-10 days and analyzed for the number of HBV genome equivalents (ge) by real time polymerase chain reaction (PCR) and for HBsAg quantity using Laurell electrophoresis. Log HBV ge/ml decreased during the follow-up from 6.8 +/- 1.1 to 5.1 +/- 1.0 to 4.2 +/- 0.8 to 3.3 +/- 1.1 (mean +/- SD). The half-life times of HBV ge increased from 1.6 days at the beginning to 4 days at the end. HBsAg decreased much slower: from 38 to 23 to 12 to 3.8 microg/ml. Half-life time was around 8 days at the beginning and 5.7 days at the end, but 11 patients showed a rapid elimination of HBsAg and HBV DNA. Hepatitis C virus (HCV) coinfection did not change the kinetics of HBV ge and HBsAg significantly. A moderate but significant suppression of HBV ge levels was observed in hepatitis D virus (HDV) coinfected patients. HBsAg levels were, however, enhanced in this cohort. In conclusion, the data suggest that expression and elimination of HBV is in most patients with acute hepatitis B not altered by coinfecting hepatitis viruses. The initial decrease of HBV ge and HBsAg in serum appears to be caused by decay or non-specific removal in the absence of replacement.     

European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty‐seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut‐off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers. J. Med. Virol. 83:95–100, 2011. © 2010 Wiley‐Liss, Inc.     

Hepatitis B virus DNA in asymptomatic HBsAg carriers: comparison with HBeAg/anti-HBe status

Sera of 17 HBeAg positive and 104 anti-HBe positive asymptomatic HBsAg carriers from two cohorts were tested for HBV DNA. HBV DNA was found in 13 of 17 HBeAg positive carriers (76.5%) and in only 7 of 104 of anti-HBe positive carriers (6.7%). Eleven of the 17 HBeAg positive carriers were retested for HBV DNA over a period of 7 to 36 months after the initial test. HBV DNA disappeared from the serum in 2 patients in spite of persistence of the HBe antigen. Of the 104 anti-HBe carriers, 89 were retested for HBV DNA over a period of 6 to 52 months after the initial test. HBV DNA disappeared from the serum in 5 of the 7 who were previously positive for HBV DNA, and persisted in 2. These findings indicate that there is an inconstant relationship between the time of seroconversion of HBeAg to anti-HBe and the disappearance of HBV DNA. In one HBeAg positive patient, HBV DNA, which was absent in the serum on first testing, was present on retesting. This suggests that the presence of HBV DNA in the serum of some patients may be intermittent. The presence of HBV DNA in the serum of some anti-HBe positive carriers accounts for the finding that they may be infective. All but one of the HBV DNA positive anti-HBe carriers were born outside North America, most in Asia. HBV DNA were found more frequently in the serum of anti-HBe positive carriers who had biochemical and histological evidence of liver disease than in carriers without such evidence.     

Molecular epidemiology of hepatitis A in St. Petersburg, Russia, 1997-2003

The molecular epidemiology of hepatitis A virus (HAV) strains circulating in the St. Petersburg and Karelia regions was studied during 1997-2003. Hepatitis A virus RNA was isolated from both clinical samples (stools or sera) and environmental samples (sewage water). RT-PCR was carried out using different primer pairs from the VP1/2A and VP1 genomic regions, the variable parts of the HAV genome. PCR products were sequenced and 306 nucleotides from the VP1/2A and 332 nucleotides from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common during the follow-up period: >90% of the isolated HAV strains belonged to that subtype. The HAV strains found in intravenous drug users belonged to subtypes IA and IIIA. Only one out of a total of 88 sequenced strains was of the IB subtype. The subtypes IB and IIIA were found only in 2001-2003, which suggests that new strains were introduced into the endemic situation. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes.     

Quasispecies nature of hepatitis C virus (HCV) in patients with chronic hepatitis C with mixed HCV subtypes

The quasispecies nature of hepatitis C virus (HCV) in patients with mixed HCV subtype infection was compared with that in patients with single HCV subtype infection. The number of HCV quasispecies was compared between 35 patients with mixed HCV subtype infection and 83 patients with single subtype infection. Subtype was determined by primers deduced from the core region and by line probe assay respectively. The number of quasispecies was evaluated by polymerase chain reaction amplification of hypervariable region 1 and by fluorescence single-strand conformation polymorphism analysis. There was no difference in clinical background between patients with mixed subtype infection and patients with single subtype infection. The number of quasispecies in patients with multiple subtype HCV infection was larger than in patients with single subtype HCV infection. The immunologic environments which allow the coexistence of more HCV quasispecies in patients with multiple HCV subtype infection differs from that in patients with single HCV subtype infection. J. Med. Virol. 54:80–85, 1998. © 1998 Wiley-Liss,Inc.