HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant

The performance of currently available hepatitis B surface antigen (HBsAg) commercial kits was analyzed by using a panel of 212 well-characterized plasma donors all over the country and a panel of nine recombinant HBsAg mutants containing single point or combinations of mutations between amino acid residues 124 and 147 of the "a" determinant. HBsAg commercial kits in this study were machine-based immunoassays with a one-step sandwich ELISA method using either an automatic closed system or manual system. The sensitivity of all machine-based assays evaluated with 105 HBsAg plasma panels was 100% (95% CL = 95.6-99.9%), whereas the specificity with 107 HBsAg negative plasma ranged from 99.07% to 100% (95% CL = 94.2-99.9%). The relative performance of these kits to detect the hepatitis B virus (HBV) mutant panel members of the "a" determinant was found to differ. Interestingly, any commercial kits with monoclonal antibody capture and polyclonal antibody detection (mono/poly), but not mono/mono Ab capture and detection, could pick up the common HBsAg Gly145Arg mutant either solely or in combination with other mutations within the "a" determinant. New versions of HBsAg test kits should recognize multiple HBsAg epitopes in order to detect mutant HBsAg, together with providing good analytical sensitivity and specificity, because of the importance of these assays in HBV diagnosis and in protecting the safety of the blood supply.     

Genetic variability of the S gene of hepatitis B virus: clinical and diagnostic impact.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergence in the entire genome of >8%, HBV genomes have been classified into eight groups designated A to H. The genotypes of HBV have distinct geographical distributions. Although preliminary clinical studies seem to indicate that there is an association between HBV genotype and natural history of infection and response to antiviral therapy, further evaluations on larger collectives of patients are necessary to give a clearer picture of the subject. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype. The influence of genotypic variability on the sensitivity of nucleic acid amplification tests (NAT) has so far been poorly investigated. Preliminary results show that new real-time NAT detect genotypes A to G with an equal sensitivity. Different mechanisms intervening at the translational or post-translational level, including conformational changes, hydrophobic changes, insertion of basic residues and reduced synthesis or secretion of HBsAg may account solely or in conjunction for escape mutations to the immune response and to detection in HBsAg immunassays. The clinical significance of S-gene mutants, needs in analogy to that of HBV genotypes, to be further investigated. HBV mutants are stable over time and can be transmitted horizontally or vertically. The sensitivity of HBsAg assays for mutant detection is continuously improved. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S-gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants and strategies for the (differential) screening of mutants need to be developed and evaluated.     

尿液中乙肝病毒DNA对HBV相关肾炎患者的诊断效果

目的 了解尿液中乙肝病毒DNA对HBV相关肾炎患者的诊断效果,为更好的诊断HBV相关肾炎(HBV-GN)提供依据.方法 选取于2013年10月～2014年10月在我院肾内科就诊的HBsAg阳性或血清HBV-DNA阳性者60例作为Ⅰ组、HBsAg阴性且清HBV-DNA阴性者60例作为Ⅱ组、同期在我院健康体检中心体检的健康者60例作为Ⅲ组.采用PCR方法检测各组研究对象尿中HBV DNA水平.结果 Ⅰ组中有38例(63.33％)患者尿HBV DNA呈阳性,Ⅱ组、Ⅲ组中无患者尿HBV DNA呈阳性.尿HBV DNA、血HBsAg和血HBeAg联合检查诊断HBV-GN的灵敏度和特异度均是最高的,其次为尿HBV DNA检测.结论 联合检测尿HBV DNA、血HBsAg和血HBeAg诊断HBV-GN效率比较高,尿HBV DNA可能作为HBV-GN的一种简单、无创、患者容易接受的辅助诊断指标.     

Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Genotyping studies show that the genetic diversity of HBV is very high even in industrialized countries. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype and could possibly lead to false negative results in samples with low-level HBsAg. It is possible that the recognition of genotypes E and F may be impaired. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity, especially for the detection of the G145R mutation and amino acid insertions or substitutions in positions 120-123. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants especially for G145R and assays for the (differential) screening of mutants need to be developed and evaluated.     

Diagnostic value of anti-HBc IgM in high HBV prevalence areas

The diagnostic value of an anti-mu-capture immunoassay for the detection of IgM antibody against hepatitis B core antigen (anti-HBc) was evaluated. Strongly positive results were obtained from the acute phase sera of the 25 acute hepatitis B patients who were hepatitis B surface antigen (HBsAg) positive and of the 18 confirmed acute hepatitis B patients who had already cleared HBsAg when symptoms developed. Negative results were obtained in 5 hepatitis A patients, 20 non-A, non-B acute hepatitis patients serologically susceptible to HBV, 22 patients with chronic hepatitis B liver disease, 15 asymptomatic HBsAg carriers, and 10 healthy patients immune from past HBV infection. Fourteen of the acute hepatitis patients remained HBsAg positive for a follow-up period of at least 6 months, and 12 of these were found consistently anti-HBc IgM negative. These were considered as chronic HBsAg carriers with a superimposed form of acute liver injury. These data show that this assay can differentiate between acute from chronic (HBsAg positive) and recent from old (HBsAg negative) hepatitis B virus infection. Thus, it should be very useful in the complex diagnostic situations encountered commonly in areas with high prevalence of HBV infections.     

Predominance of genotype A HBV in an HBV-HIV-1 dually positive population compared with an HIV-1-negative counterpart in Japan

Hepatitis B virus (HBV) has seven genotypes, A to G. Previous studies have shown that genotype C is the most prevalent strain in chronic HBV carriers in East Asia. This study was undertaken to investigate the epidemiology of HBV genotypes among Japanese patients who are coinfected with human immunodeficiency virus type 1 (HIV-1). The sequences of the complete hepatitis B surface antifen (HBsAg) genes were obtained from 18 coinfected Japanese patients. Among the 18 patients, 12 of 13 men who had sex with men (MSM) had genotype A (92%), whereas only one of five heterosexual or hemophiliac patients had genotype A. The predominance of genotype A HBV in MSM showed a striking contrast to the current genotype prevalence in the Japanese population. Owing to the recent decrease in the rate of vertical transmission in Japan, the role of sexual behavior in the transmission of HBV cannot be overestimated. Thus, the relative proportion of genotype A may gradually increase in Japan.     

Occult HBV infection

The long-lasting persistence of hepatitis B virus (HBV) genomes in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for the HBV surface antigen (HBsAg) is termed occult HBV infection (OBI). Although in a minority of cases the lack of HBsAg detection is due to infection with variant viruses unrecognized by available assays (S-escape mutants), the typical OBI is related to replication-competent HBVs strongly suppressed in their replication activity. The causes of HBV suppression are not yet well clarified, although the host’s immune surveillance and epigenetic mechanisms are likely involved. OBI is a worldwide diffused entity, but the available data of prevalence in various categories of individuals are often contrasting because of the different sensitivity and specificity of the methods used for its detection in many studies. OBI may have an impact in several different clinical contexts. In fact, it can be transmitted (i.e., through blood transfusion and liver transplantation) causing classic forms of hepatitis B in newly infected individuals. The development of an immunosuppressive status (mainly by immunotherapy or chemotherapy) may induce OBI reactivation and development of acute and often severe hepatitis. Finally, evidence suggests that OBI can favor the progression of liver fibrosis, in particular in HCV-infected patients. The possible contribution of OBI to the establishment of cirrhosis also implies its possible indirect role in the development of hepatocellular carcinoma. On the other hand, OBI may maintain most of the direct transforming properties of the overt HBV infection, such as the capacity to integrate in the host’s genome and to synthesize pro-oncogenic proteins.     

False-Negative Hepatitis B Virus (HBV) Surface Antigen in a Vaccinated Dialysis Patient with a High Level of HBV DNA in the United States

Screening with hepatitis B surface antigen (HBsAg) is highly recommended for at-risk individuals. Mutations in the HBsAg can result in an inability to detect the virus during routine screening. We describe a hemodialysis patient found to have high levels of hepatitis B virus (HBV) DNA and HBV antibody but negative HBsAg on two routine assays.     

Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing.

The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk.     

乙型肝炎病毒表面大蛋白检测用于筛查隐匿性HBV感染

目的 用血清学方法检测乙型肝炎病毒表面大蛋白(HBLP)来筛查隐匿性HBV感染,探讨临床隐匿性HBV感染的血清学检测策略.方法 在日常工作中从临床榆测备份管随机收集2000份用国产ELISA试剂检测HBsAg阴性结果的血清标本,双份分装-20℃冻存,单份标本实施HBLP检测,HBLP阳性标本增加另外两种共三种国产ELISA试剂双份复查HBsAg;HBLP阳性标本再经美国超灵敏MONOLISA HBsAg ULTRA试剂双份检测HBsAg,并行双份DNA定量分析、结果取均值.结果 2000份HBsAg阴性标本共检出15例HBLP阳性:HBLP阳性标本用三种国产ELISA试剂双份复查HBsAg结果一致阴性;通过美国超灵敏MONOLISA HBsAS ULTRA试剂复查HBsAg结果全部阳性;前述15份标本HBV DNA定量分析结果显示均小于500拷贝/ml,其中400～500拷贝/ml 1例,300～400拷贝/ml 3例,200～300拷贝/ml 5例,100～200拷贝/ml 4例,小于100拷贝/ml 2例.结论 用国产常规ELISA试剂检测HBsAg普遍存在漏检,漏检标本HBLP结果可能阳性,检测HBLP有利于筛查出隐匿性H BV感染,为积极寻找临床隐匿性HBV感染的检测策略提供了血清学参考.     

隐匿性乙型肝炎病毒感染检测的实验研究

目的 了解乙型肝炎病毒表面抗原（HBsAg）阴性个体HBV DNA检出情况,探讨检测乙型肝炎病毒表面大蛋白（HBLP）用于筛查临床隐匿性HBV感染的血清学检测策略.方法 根据日常检测乙型肝炎病毒血清学模式（HBVM）结果,将HBsAg阴性标本分为乙型肝炎病毒表面抗体（HBsAb）阳性和阴性两大类,除外HBVM抗原抗体同时阳性等特殊模式,从临床检测备份管随机收集各1000份共计2000份血清标本,双份分装-20℃冻存;用多管混合的方法实施HBV DNA定量分析,筛选出阳性标本;HBV DNA阳性标本行HBLP检测,再经美国超敏MONOLISA HBsAg ULTRA试剂复检HBsAg.结果 1000份HBsAb阳性标本未检出HBV DNA阳性;1000份HBsAb阴性标本共检出19例HBV DNA阳性;19份HBV DNA阳性标本经HBLP检测和美国超敏试剂复检HBsAg均呈阳性.19份HBV DNA定量分析结果显示：大于500拷贝/ml 2例,400～500拷贝/ml 3例,300～400拷贝/ml 3例,200～300拷贝/ml 7例,100～200拷贝/ml 4例.结论 用国产ELISA试剂常规检测HBsAg漏检标本多来自HBsAb阴性个体;漏检个体HBLP结果可能阳性,检测HBLP有利于筛查隐匿性HBV感染;本研究为积极寻找临床隐匿性HBV感染的检测策略提供了血清学参考.     

Detection of HBV DNA by nested-PCR in a HBsAg and anti-HBc negative blood bank donor.

BACKGROUND: The criteria and protocol adopted in serological screening of blood bank donors have significantly reduced the possibility of HBV transmission. However, it is possible that, in a very recent phase of HBV infection, HBsAg seronegative donors be able to transmit the virus. This study reports a case of a donor with this serological profile who was involved in the viral transmission to a seronegative receptor. CASE REPORT: A blood donor had her sample tested for HBsAg and anti-HBc, which resulted negative. At the second donation the sample demonstrated to be seropositive for anti-HBc, anti-HBs and seronegative for HBsAg. The first stored sample was tested for the presence of HBV DNA. Two fragments could be identified in the genomic region corresponding to HBV core and precore. Only one individual was involved in the transfusion of hemo-derivatives originating from the processing of this bag, and was seropositive for HBsAg, HBeAg and anti-HBc markers and seronegative for the anti-HBe and anti-HBs markers. CONCLUSION: This case illustrates the possibility of the occurrence of HBV transmission from blood bank donors seronegative for HBsAg and anti-HBc. This fact could be associated with the possibility of the donor to be in the pre-seroconversion phase of a recent infection, when the levels of HBsAg present in the circulation are below the limits of detection. The implementation of molecular tests or higher sensitivity HBsAg assays could further reduce the risk of HBV transmission via blood transfusion.     

Hepatitis B virus (HBV) vaccine-induced escape mutants of HBV S gene among children from Qidong area, China

Hepatitis B virus (HBV) infection is the main factor, which induces hepatocellular carcinoma (HCC) in Qidong high-risk area, China. To prevent HBV infection is the most important strategy to inhibit the HCC carcinogenesis. A large project was performed in Qidong area to protect newborn babies from the HBV infection that 80,000 children born between 1984 and 1990 were vaccinated. After three times of follow-up studies, 15 screened children were found to have symptoms of illness showing persistent elevation of serum glutamic-pyruvic transaminase (ALT). From these previously collected data, we found that the ALT levels of five vaccinees with negative hepatitis B surface antigen (HBsAg) were significantly higher than those of 10 vaccinees with positive HBsAg. Furthermore, with the passage of time, the difference of ALT levels between the two groups (HBsAg negative and positive groups) diminishes. After cloning and sequencing of the HBsAg "a" epitope coding sequences, we found that mutations in "a" epitope were correlated with the absence of detectable anti-HBsAg, while no mutations were seen in the anti-HBsAg positive infections. We also found that majority of point mutations were occurred in the coding sequences of the first loop structure in "a" epitope. The structure of double loop conformation in "a" epitope was conservative, and important for HBV antigenicity. These changes in a double loop conformation would escape neutralization by vaccine-induced antibody.     

Should HBV DNA NAT replace HBsAg and/or anti-HBc screening of blood donors?

Prevention of transfusion-transmitted hepatitis B virus (HBV) has historically relied on serological screening of blood donors using progressively more sensitive HBsAg assays; in some countries anti-HBc assays have also been employed to detect chronic carriers with low-level viremia who lack detectable HBsAg. Nucleic acid amplification testing (NAT) for HCV and HIV has been successfully introduced to screen donors in many developed countries over the past several years; for logistical and cost reasons HCV/HIV NAT screening has been applied to mini-pools (MP) of eight to 96 donor specimens, with only minimal impact of MP dilutions on clinical sensitivity for interdiction of window period (WP) donations. In several countries (e.g., Japan and Germany), HBV NAT has been added to HIV/HCV MP-NAT blood donor screening with small incremental yields of HBsAg/anti-HBc-negative donations, and the major vendors of NAT systems (Roche and Chiron/Gen-Probe) have been developing triplex assays that include HBV DNA detection capacity without compromising HIV or HCV detection. Pooled specimen HBV NAT has also become the standard of practice for screening source plasma donors, with pressure to include HBV DNA detection as a required procedure for use of recovered plasma in manufacture of fractionated derivatives. However, there is controversy over the magnitude of the incremental yield and clinical benefit of HBV MP-NAT over serological screening strategies, as well as the impact of implementation of HBV NAT on need for retention of HBsAg and anti-HBc screening. This presentation will review recent modeled and empirical data on the value of HBV MP- and individual donation (ID)-NAT for detection of (1) pre-HBsAg WP units and (2) chronic anti-HBc-reactive carriers with undetectable HBsAg. The presentation will also review policy considerations and data that address the potential for discontinuation of either HBsAg or anti-HBc following implementation of HBV NAT. Finally it will address the cost effectiveness of incorporation of HBV DNA detection into HBV screening and NAT testing algorithms.     

Detection of HBsAg mutants

HBsAg screening is carried out routinely to detect hepatitis B virus (HBV) infection. The immunoassays used employ capture antibodies often having specificity for epitopes present on the antigenic (a) determinant of the HBsAg. Loss of detection may occur due to mutations within and/or outside of the a determinant that affect conformational epitope recognition or HBsAg secretion or expression. Most of the mutations associated with immune escape occur within the second loop of the a determinant. In order to detect these HBsAg mutants, antibodies to subdominant regions within the a determinant or outside of the HBsAg may be required, and this has been the focus of many recent studies. Any changes to immunoassay formulations should also address the possible effect of HBV genotypic polymorphisms on assay specificity and sensitivity. HBsAg mutants may also be identified through nucleic acid detection of HBV in serum. Various molecular analysis methods have been developed to provide specific and sensitive detection of HBsAg mutants, including sequencing, limiting dilution cloning PCR (LDC-PCR), gap ligase chain reaction (gLCR), and real time PCR. Sequencing the HBsAg coding region provides specific information on the nucleotide sequence; however, it is relatively insensitive for the detection of minority quasispecies. Other nucleic acid methods offer greater sensitivity for the detection of point mutations. To improve immunoassays, further research will be required to increase detection sensitivity and specificity. Ultimately, a better understanding of the structure of antibody-bound HBsAg will help identify the immunological targets required for the accurate detection of HBsAg in blood.     

Transmission of hepatitis B virus (HBV) genotypes among Japanese immigrants and natives in Bolivia

Hepatitis B virus genotypes are associated with transmission pattern, virological and clinical features and outcome of the chronic infection course. HBV genotypes other than Genotype F (HBV/F) are considered a reflection of human migration into South America. A total of 487 individuals in Bolivia, including Japanese immigrants (n=287) and natives (n=200), were screened for HBV serological markers. Overall 22/487 (4.5%) of the subjects were positive for HBsAg, 217/487 (44.5%) for anti-HBc and 162/487 (33.3%) for anti-HBs. Genotypes were determinable in 22 cases by EIA, followed by sequencing and phylogenetic analysis in 17 cases. HBV genotype distribution in Japanese and Bolivians was HBV/F (4 and 8); HBV/C (5 and 3); and HBV/B (1 and 1), respectively. Phylogenetic analyses of nine complete and eight partial (HBsAg/pre-core/core region) genomes, revealed that HBV/F strains cluster with previously reported regional strains, whereas HBV/B and HBV/C strains belonged to Asian subgenotype B2 (Ba) and C2 (Ce), respectively. Japanese immigrants might have introduced HBV/B and HBV/C to natives in Bolivia, conversely, exposed to the indigenous HBV/F. This report provides evidence of an inter-communities transmission of HBV revealed by its genotypes. Further study is required to investigate peculiarities of the genotypes in different ethnic groups in Bolivia.     

Occult hepatitis B in the genotype H‐infected Nahuas and Huichol native Mexican population

Mexico is considered to be a low endemic country for HBV infection. However, a high anti‐HBc against a low hepatitis B surface antigen (HBsAg) seroprevalence is the reported characteristic of native Mexicans. HBV diagnosis and genotype distribution was examined in native populations (Nahuas and Huichol, n = 306), and compared to a non‐native population (Mestizos, n = 17). Overall, 6% of the natives were positive for HBsAg and 33% had detectable anti‐HBc. HBsAg prevalence was lower in Nahuas compared to Huichols (1.4% vs. 9.4%, P < 0.002). Occult hepatitis B was detected in 14.2% (41/289) of natives, who either tested positive (5.88%, 17/289 HBsAg‐negative) or negative for anti‐HBc marker (8%, 24/289 HBsAg‐negative). Age‐adjusted anti‐HBc seroprevalence and HBsAg quantitation revealed a sub‐optimal sensitivity of conventional immunoassays. Nahuas had HBV/H and Huichol had HBV/A as the predominant genotypes followed by genotypes D, C, B, A, and D, G and H, respectively. A less variable HBV/H was characteristic in Mestizos, compared to a much variable HBV/H identified among the Nahuas. In conclusion, these findings indicate a high HBV endemicity among native Mexican groups where occult B infection is common. The different distribution of HBV genotypes among natives suggests multiple reservoirs of HBV from which these genotypes spread into the local communities. High anti‐HBc seroprevalence against a low HBsAg prevalence rate may be due to the limited sensitivity of the immunoassays for the detection of HBsAg that are available in Mexico and/or unknown immunogenetic characteristics of native Mexicans. J. Med. Virol. 82:1527–1536, 2010. © 2010 Wiley‐Liss, Inc.     

HIV抗体酶联免疫诊断试剂检测不同基因型抗体的研究

目的 分析不同HIV抗体酶联免疫诊断试剂对检测HIV不同基因型抗体的情况。方法 对不同地区的20份HIV抗体阳性样品中HIV核酸进行扩增，对PCR产物进行测序并进行基因型别分析。用不同试剂对系列稀释的不同基因型样品进行检测。结果 20份样品均为HIV RNA阳性，其中9份样品为HIV B亚型，9份样品为：HIV C或BC重组，2份为HIVAE重组。不同试剂对HIV不同基因型抗体的检测灵敏度无明显差异。结论 我国主要的商业化HIV抗体诊断试剂产品检测不同基因型抗体的能力无明显差异。     

The relation between virus genotypes and coexisting surface antigen and antibody in patients with hepatitis B, and the development of preS region deletions

In recent years, there has been renewed interest in hepatitis B virus (HBV) genotypes. Our previous data have shown the importance of in-frame deletions in the preS region in cases of coexisting hepatitis B surface antigen (HBsAg) and anti-hepatitis B surface antibody (HBsAb). The aim of the present study was to investigate the relation between HBV genotypes and coexisting HBsAg and HBsAb, preS deletion mutants. We investigated the HBV genotypes in 9 patients with coexisting HBsAg and HBsAb. Viral DNA was extracted from the patients' sera and the HBV S gene region was amplified by polymerase chain reaction (PCR). HBV genotypes were then investigated by restriction fragment length polymorphism(RFLP) analysis. All 9 cases were found to have genotype C. This result clearly indicates that the unique finding of coexisting HBsAg and HBsAb depends on the HBV genotype. After genotypic screening was performed for HBV-positive samples from randomly selected 60 cases. The results of the 60 cases we investigated showed 26 cases of genotype B (43.3%), 31 cases of genotype C (51.7%), 1 case of coexisting genotype B and C (1.7%), and 2 cases of other genotypes (3.3%). Of the 60 cases, 45 cases consisting of 21 with genotype B and 24 with genotype C were subject to direct DNA sequencing of PCR products in the preS region to determine the presence or absence of preS deletion mutants. PreS deletion mutants were found in a total of 7 of the 45 HBV cases that underwent sequencing(7/45; 15.6%), and 6 of these had genotype C (6/24 cases, 25.0%), whereas only 1 had genotype B (1/21 cases, 4.8%). These results demonstrate a greater frequency of preS deletion mutants with genotype C. Interestingly, many preS deletion mutants showed deletions at the same point, namely the amino terminal side of the preS2 region. These results indicate that the HBV genotype is involved in the molecular pathogenesis of hepatitis B.