山东地区HBV基因型分布及拉米夫定抗病毒治疗对基因分型的影响

目的 确定乙型肝炎病毒（HBV）基因型在山东地区的分布及拉米夫定抗病毒疗效的影响。方法 采用PCR微板核酸分子杂交酶联显色技术，对临床服用拉米夫定一年的131例乙型肝炎患者血清中的HBV．DNA进行基因分型，与未服用拉米夫定组对照。结果 治疗组C型为87．4％（90／103），D型为12．6％（13／103）。对照组C型为89．3％（25／28），D型为10．7％（3／28），两组间差异无统计学意义。均未发现A、B、E和F基因型。结论 首次从临床慢性乙肝患者中得出山东地区HBV优势基因型为C基因型，同时存在D基因型。服用拉米夫定治疗一年对HBV基因型没有影响。     

Correlation between pretreatment viral sequences and the emergence of lamivudine resistance in hepatitis B virus infection

The emergence of amino acid or nucleotide substitutions leads to lamivudine resistance in hepatitis B virus (HBV) infected patients. The aim of this study was to investigate whether viral sequences help predict the emergence of lamivudine resistance. The study subjects comprised 59 consecutive patients infected with HBV treated with daily therapy of 100 mg lamivudine. Among those, 32 patients with adequate pretreatment serum preservation were investigated for the correlation between viral amino acid substitutions and the appearance of lamivudine resistance with consideration of clinical background by determining dominant HBV full open reading frames. Viral resistance to lamivudine emerged in 28 of 59 patients (47%) in a median period of 2.45 years. Sequence comparisons of HBV genomes between patients who later developed lamivudine resistance and patients who did not revealed the existence of significant differences between the two groups in the pre‐S1 84 (P = 0.042), pre‐S2 1 (P = 0.017) and 22 (P = 0.015), and polymerase tp 95 (P = 0.046), judged by a log‐rank test. Viral sequence analyses revealed the presence of amino acid substitutions in HBV pre‐S1 and pre‐S2 that may be associated with the emergence of lamivudine resistance during chronic HBV infection. J. Med. Virol. 84:1360–1368, 2012. © 2012 Wiley Periodicals, Inc.     

High frequency of lamivudine resistance mutations in Brazilian patients co‐infected with HIV and hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co‐infected with HIV and hepatitis B virus (HBV). A cross‐sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an “in‐house” real‐time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty‐eight patients with co‐infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L + rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sI195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481–1488, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular epidemiology of hepatitis B, C and D viruses in Turkish patients

Summary. Different genotypes of the hepatitis viruses may influence the clinical outcome of the disease. The distribution of genotypes may vary according to geographical regions. The aim of this study was to evaluate hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV) genotypes in Turkish patients with chronic hepatitis in a large cohort of patients. Genotyping was performed in 41, 59 and 365 patients with chronic hepatitis B, D and C, respectively, and 36 hemodialysis patients with chronic hepatitis C. Genotypes were determined by direct sequencing in hepatitis B and by polymerase chain reaction-restriction fragment length polymorphism in hepatitis C and D patients. In addition, HBV subtyping by multiplex PCR and subtype specific ELISA were performed in 83 and 71 HBsAg (+) blood donors, respectively. All hepatitis B (100%) and hepatitis D (100%) patients had genotype D and type I, respectively. HBsAg subtyping by two methods yielded that 99% of the patients were subtype ayw. S gene amino acid sequence in the 41 patients included for HBV genotyping revealed the ayw2 subtype. Genotype distribution of 365 patients with chronic C hepatitis were as follows: 306 (84%) patients genotype 1b, 43 (11%) patients genotype 1a, 10 (3%) patients genotype 2, 3 (1%) patients genotype 3, 3 (1%) patients genotype 4. Among 36 patients receiving hemodialysis, 28 (78%) patients had genotype 1b and 8 (22%) patients had genotype 1a. The study indicates that Turkish patients with chronic viral hepatitis show very little genotypic heterogeneity. Subtype ayw and the genotype D of HBV DNA, and the type I of HDV RNA represent almost 100% of related infections. The genotype 1b of HCV RNA was found to be significantly dominant in Turkish patients.     

Identification and characterization of novel hepatitis B virus subgenotype C10 in Nusa Tenggara, Indonesia

Six novel subgenotypes (B7, B8, C6, C8, C9, and D6) within three hepatitis B virus (HBV) genotypes (B–D) were recently identified in Indonesia. To further characterize HBV in this country, 18 HBV-viremic samples obtained from blood donors in Nusa Tenggara, Indonesia, were subjected to phylogenetic analysis of an 1.6-kb partial or full-length sequence. Thirteen HBV isolates were classified into genotype B with four distinct subgenotypes [B3 (n = 2), B5 (n = 1), B7 (n = 4), and B8 (n = 6)], followed by 4 HBV isolates of genotype C (HBV/C); the remaining one isolate was of D (D1). As for the four HBV/C isolates, one isolate segregated into subgenotype C1, and two into C2. The remaining HBV/C isolate [C0901177(NT3)] differed from reported HBV/C isolates (C1–C9) by 4.6–7.7% over the entire genome and did not show evidence of recombination with any of the known HBV genotypes/subgenotypes, justifying its conclusive assignment into a novel subgenotype (C10) within genotype C.     

Hepatitis B virus genotypes and lamivudine resistance mutations in HIV/hepatitis B virus-coinfected patients

BACKGROUND: Differences in subtypes, hepatitis B early antigen (HBeAg)-negative variants, and drug resistance mutations all seem to influence the clinical and therapeutic outcome in patients with chronic hepatitis B virus (HBV) infection. Information available on the prevalence and distribution of distinct HBV variants in HIV-positive patients is scarce. METHODS: All HIV-infected patients with persistent serum hepatitis B surface antigen and detectable HBV viremia were identified in a reference HIV clinic located in Madrid, Spain. HBV load, subtypes, precore (PC) and basal core promoter (BCP) variants, and lamivudine (LAM) resistance mutations were analyzed. RESULTS: A total of 81 HBV/HIV-coinfected patients (4.1%) were identified in a population of 1968 HIV-positive patients. Plasma specimens with detectable HBV viremia could be obtained from 62 subjects, and this was the study population that underwent further virologic characterization. HBV genotype distribution was as follows: A (n = 27), D (n = 27), E (n = 1), F (n = 2), and G (n = 3). Two patients had mixed HBV genotypes (A/E and A/F). HBV subtype A was predominant (74%) among patients infected through sexual contact, whereas HBV-D was most frequent (74%) among intravenous drug users (P < 0.001). PC/BCP mutants were more frequent in patients with HBV-D than in those with HBV-A (63% vs. 18%; P < 0.01). Median time on LAM was 40 months; patients with HBV-A tended to show LAM resistance mutations more often (53% vs. 44%) and to develop them earlier (35 vs. 45 months) than patients with HBV-D. The dual L180M + M204V/I mutant was the predominant resistance pattern, although a triple rt173V + 180M + 204V, which acts as a vaccine escape mutant, was found in 1 individual. In the multivariate analysis, patients with LAM resistance mutations were significantly more frequently HBeAg-positive and older than individuals with wild-type HBV. Hepatitis-delta was recognized in 13 (21%) of these 62 HBV viremic patients, with no association with specific HBV variants. CONCLUSION: Risk transmission group, age, and positive serum HBeAg are the main determinants of distinct HBV virologic variants, including HBV genotypes and LAM-resistant mutants, in HBV/HIV-coinfected patients.     

Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland

Hepatitis B virus (HBV) infection is one of the major global epidemiological problems. The aim of our study was to determine the distribution of HBV genotypes in Poland since the data concerning the spread of HBV viruses in the central-eastern region of Europe is still very limited. HBV DNA was extracted from 58 serum samples. To quantify the level of HBV DNA the Roche Amplicor HBV Monitor Assay was used. To genotype and assign HBV subtypes DNA sequencing methods were performed. The HBV virus from 43 serum samples from hepatitis B infected patients was genotype A (74.1%), 12 cases had genotype D (20.7%), and 3 had the rare in Europe genotype F (5.2%). Prediction of HBV serological subtypes based on HBsAg sequencing showed almost 100% occurrence of subtype adw2 in the group of genotype A samples, three different subtypes in genotype D (ayw2, ayw3, and ayw4), and equal distribution of subtype adw4q- in all 3 cases of genotype F, also the most prevalent subtype in the Amerindians. Our results coincide with the general European HBV prevalence. However, HBV genotype F, which is not a common genotype in European countries, was detected and so was relatively high occurrence of genotype D, which may reflect historical and ethnical migration events in Poland in the past.     

Naturally occurring MHR variants in Turkish patients infected with hepatitis B virus

Major B-cell epitopes are located at the major hydrophilic region (MHR) of hepatitis B virus (HBV) surface antigen (HBsAg). The genotypes, subtypes, and naturally occurring amino acid (aa) substitutions of MHR were analyzed in 81 Turkish adult patients (41 inactive HBsAg carriers and 40 patients with chronic hepatitis B) by direct sequencing of the S gene fragment. All the isolates were genotype D according to the phylogenetic analysis. The most common HBsAg subtype was ayw2, followed by ayw3 while one isolate specified ayw4 by encoding Leu127. MHR variants were detected in 22 of the 81 (27.2%) isolates. The prevalence was significantly higher in the chronic hepatitis B group (42.5%) compared to inactive HBsAg carriers (12.2%). Twenty-two samples had a total of 26 amino acid substitutions involving 14 positions. The majority of the patients had a single variation. Most of the amino acid substitutions were located at the HBs1 region of the MHR, while 9 of the 26 were in the classic "a" determinant (aa 124-147). When samples with "a" variants were evaluated by two different commercial HBsAg tests, only the isolate with Ser143Leu variation had a decreased reactivity in the assay using monoclonal antibodies for capture and detection. In conclusion, the findings of the study was in accordance with previous studies showing HBV genotype and subtype homogeneity (genotype D/ayw) in Turkey. Naturally occurring MHR and "a" determinant variants were common, especially among chronic hepatitis B patients. The influence of detected "a" variants on diagnostic assays was limited.     

乙型肝炎病毒基因分型及临床应用研究

目的了解常州地区乙型肝炎病毒基因型分布特征,探讨其基因型与肝功能损伤、病毒复制水平及对拉米夫定疗效的关系. 方法采用巢式聚合酶链反应 (nest-PCR), 扩增乙型肝炎病毒S基因区, 用末端标记方法对PCR产物标记并直接测序, 测序结果和GenBank中登录的标准基因型序列相比较. 结果对该地区146份不同HBV感染者血清HBV DNA进行了基因分型,B型51份 (34.9%),C型95份(65.1%),未发现B、C以外其他基因型;丙氨酸转氨酶(ALT)水平分别为383.8±335.7IU和364.3±333.7 IU,(t=0.335,P>0.05)、HBV DNA含量分别为107.795±1.22和107.69±1.19拷贝/毫升(t=0.138,P>0.05)、HBeAg 阳性数分别为36/51和64/95,(χ2=0.159,P>0.05);104例慢性乙型肝炎中B型为43例、C型为61例,28例肝硬化和肝癌患者检出B型4例、C型24例,二组比较χ2=7.65,P<0.01;23例B基因型患者和45例C基因型患者接受48周以上拉米夫定治疗,48周后反跳者B型为18例,C型为14例,χ2=13.49,P<0.001.结论本地区HBV DNA基因型为B型和C型;二种基因型丙氨酸转氨酶水平、病毒复制水平和HBeAg表达水平差异均无显著性;C基因型与肝硬化和肝癌关系密切;拉米夫定对C基因型患者的疗效强于B型.     

Clinical course and predictive factors of virological response in long-term lamivudine plus adefovir dipivoxil combination therapy for lamivudine-resistant chronic hepatitis B patients

The aims of this study were to assess the long‐term efficacy of lamivudine (LAM) plus adefovir dipivoxil (ADV) combination therapy in patients with chronic hepatitis B resistant to LAM, to identify predictive factors of complete viral response (HBV‐DNA <2.6 log copies/ml at 12 months of combination therapy), and to analyze amino acid substitutions associated with treatment resistance in the hepatitis B virus (HBV) genome. Seventy‐two patients who received ADV in addition to LAM for breakthrough hepatitis were enrolled. Undetectable HBV‐DNA was observed in 61%, 74%, 81%, 84%, and 85% at 12, 24, 36, 48, and 60 months of combination therapy, respectively. On multivariate analysis, undetectable HBV‐DNA during the preceding LAM monotherapy (P < 0.0001), alanine aminotransferase value ≥ the upper limit of normal × 6 (P = 0.006) and HBV‐DNA level < 6.0 log copies/ml at the initiation of combination therapy (P = 0.007) were independent significant predictors of complete viral response. The cumulative rate of undetectable HBV‐DNA was significantly higher in patients with response to the preceding LAM monotherapy than in those with poor response to it. Breakthrough hepatitis occurred in three patients without complete viral response and with poor response to the preceding LAM monotherapy, and rtA181A/V substitution was detected in one of the three patients. In conclusion, undetectable HBV‐DNA during the LAM monotherapy was the strongest independent predictor of complete viral response to the following combination therapy. The efficacy of LAM plus ADV combination therapy may be determined by viral response to the preceding LAM monotherapy. J. Med. Virol. 83:953–961, 2011. © 2011 Wiley‐Liss, Inc.     

拉米夫定治疗中乙型肝炎病毒基因序列变异特点及其准种研究

目的研究拉米夫定治疗中乙型肝炎病毒（HBV）逆转录酶区核苷酸序列变异、特点、含量及其与基因型和病毒载量的关系。方法普通DNA测序法检测拉米夫定治疗的117份慢乙肝患者血清HBV逆转录酶区基因序列及其基因型;其中99份用TaqMan法定量HBVDNA;64份用焦磷酸测序（Pyrosequencing）检测YMDD基序中碱基的频率。结果HBVYMDD变异组中C型43例,B型10例,A／B混合型1例;YMDD不变异组中C型54例,B型8例,D型1例,HBVDNA含量平均对数值在变异组和不变异组分别为：6．5699和6．6165;YMDD变异与其基因型及病毒载量无统计学意义;rtL180M位点变异与rtM204I／V位点变异高度相关;并且HBV野生株与变异株均同时存在。结论结合两种测序方法可以用来研究拉米夫定治疗中HBV基因序列变异情况及对YMDD耐药株进行定量。     

Response to long-term lamivudine treatment in patients infected with hepatitis B virus genotypes A, B, and C

Response to lamivudine treatment longer than 1 year was compared in 15 patients persistently infected with hepatitis B virus (HBV) genotype A, 38 with genotype B, and 449 with genotype C. Patients with genotype A were younger (median age 37 [range 24-49] vs. 47 [24-67] or 44 [18-73], P = 0.015), possessed hepatitis B e antigen (HBeAg) more frequently (73% vs. 21% or 56%, P < 0.001) and HBV DNA in higher levels (8.6 [6.1-8.7] vs. 6.5 [<3.7-8.7] or 6.5 [<3.7-8.7] log genome equivalents (LGE)/ml, P = 0.024) than those with genotype B or C. During lamivudine, YMDD mutants (89% vs. 53% or 42%, P = 0.0001) and breakthrough hepatitis developed more often (47% vs. 21% or 29%, P = 0.023) in patients with genotype A than B or C. YMDD mutants elicited more frequently in patients with genotype A than B or C who were positive (82% [9/11] vs. 25% [2/8] or 48% [117/245], P = 0.037) or negative for HBeAg (75% [3/4] vs. 30% [9/30] or 33% [68/204], P = 0.003). HBeAg (hazard ratio 2.1 [95% confidence interval 1.53-2.92], P < 0.001) and genotype A (2.78 [1.08-7.12], P = 0.034) enhanced the emergence of YMDD mutants by the Cox proportional hazard model. The risk for breakthrough hepatitis was increased by the baseline alanine aminotransferase level <500 IU/L (2.56 [1.82-5.50], P = 0.018), HBeAg (2.11 [1.40-3.16], P < 0.001), cirrhosis (1.92 [1.24-2.97], P = 0.004) and HBV DNA > or =8.0 LGE/ml (1.57 [1.04-2.36], P = 0.03); it was influenced by genotypes only in patients with HBeAg. In conclusion, HBV genotypes help in predicting response to long-term lamivudine treatment and development of YMDD mutants in patients with chronic hepatitis B.     

Do different lamivudine‐resistant hepatitis B genotypes carry the same risk of entecavir resistance?

Entecavir switch is one of the treatment options for lamivudine‐resistant hepatitis B (HBV) patients in Asia. This study examined the outcome of patients with different baseline resistance genotypes in a cohort study. In this study, 14 patients with chronic HBV were treated with entecavir 1 mg/day for 5 years. Enrolment criteria include: documented lamivudine resistant mutations, treatment with adefovir 10 mg/day for at least 24 weeks, and Child‐Pugh score <7. Most had previous failed adefovir therapy and compensated cirrhosis of the liver. Clinical outcomes, liver biochemistries, and HBV DNA were monitored regularly. Patients with virologic breakthrough were rescued with add‐on adefovir. At the end of the treatment period, the mean HBV DNA fell from 5.92 × 10⁶ (baseline) to 3.67 × 10¹ IU/ml. The presence of a HBV polymerase rtM204V mutation at the baseline was found to be the major risk factor for adverse outcomes. Compared to the patients with the rtM204I mutant, patients with the rtM204V mutant had increased risk of virologic breakthrough (80% vs. 0%, P = 0.010) requiring add‐on adefovir, slower virologic responses (log rank test, P = 0.0011), failure to reach undetectable HBV DNA levels (60% vs. 0%, P = 0.045), and higher risk of entecavir‐resistance (60% vs. 0%, P = 0.045). All the patients with rtM204I and rtA181 mutants had undetectable HBV DNA from 18th month. In summary, lamivudine‐resistant HBV patients with the rtM204V mutation have the highest risk of developing entecavir resistance, and entecavir monotherapy should be avoided. Those with the rtM204I and rtA181V mutations may have lower risks, but regular surveillance for viral breakthrough is required. J. Med. Virol. 85:26–33, 2012. © 2012 Wiley Periodicals, Inc.     

Significance of hepatitis B virus genotypes A to E in a cohort of patients with chronic hepatitis B in the Seine Saint Denis District of Paris (France)

The aim of this study was to examine the genetic variability of the hepatitis B virus (HBV) and its significance. HBV genotypes, core promoter and precore mutants were characterized in 109 consecutive patients with biopsy-proven HBV chronic hepatitis. Genotypes A (26.6%), B (12.8%), C (18.3%), D (18.3%), and E (14.7%) indicate a wide genotypic distribution. Patients were from Asia (30.3%), Europe (28.4%), Sub Saharan Africa (23.9%), the Caribbean (10.1%), North Africa (5.5%), and Madagascar (1.8%). HBV genotypes A and D (HBV/A and /D) infected all subgroups except Asian patients. HBV/B or /C were found in 97% of Asian patients, whereas HBV/E only infected sub-Saharan African and Caribbean patients. Differences according to genotypes were: an increased prevalence of anti-HBe antibodies in patients infected with HBV/D (P = 0.003), higher serum transaminases in patients infected with HBV/A and/D (P = 0.043), more severe liver fibrosis in patients infected with HBV/A, /C and/D (P = 0.02). Precore and core promoter mutants were found in 87% of anti-HBe positive patients, and were associated with HBV/D (P = 0.04) and severe liver fibrosis (P = 0.002). It is concluded that HBV genotypes A, B, C, D, and E circulate in the Seine Saint Denis District, reflecting the geographical origin of patients. HBV/A, /C and/D seem to be associated with more severe hepatic disease.     

Hepatitis B virus surface gene mutants in immunoprophylaxis-failed infants from Southern China

Hepatitis B virus (HBV) vaccination was considered the most powerful tool for prevention of HBV transmission from mother to infant. In 1992, a universal HBV vaccination program for infants was launched in China. The aim of this study was to investigate the HBV surface antigen (HBsAg) variations in immunoprophylaxis-failed infants, vaccine free infant patients and adult chronic hepatitis B (CHB) patients (without nucleoside analog treatment). Immunoprophylaxis-failed infants were recruited from two centers (Guangdong and Chongqing, representing Southern China). HBV serum markers, including HBsAg, hepatitis e antigen (HBeAg), and core antigen, were detected by the enzyme-linked immunosorbent assay. HBV DNA load was detected by quantitative polymerase chain reaction. Nucleotide sequences encoding HBsAg were amplified and analyzed. Frequencies of amino acid substitutions within the second loop of “a” determinant (region with greater local hydrophilicity) in immunoprophylaxis-failed infants were clearly higher than the unvaccinated infant patients and adult CHB patients (9.6% vs 0% vs 3.8%; χ ² = 7.454; P = 0.024). More than 50% (52.8%) aa substitutions in immunoprophylaxis-failed infants were located in B cell epitopes, while 64.6% aa substitutions in adult CHB patients were located in CTL cell epitopes. HBeAg negative patients had higher substitution frequency in CTL and Th cell epitopes, and in immunoprophylaxis-failed infant patients with substitution in major hydrophilic region region had a higher alanine aminotransferase level (68.6 ± 111.5 vs 62.1 ± 132.2; P = 0.026), and lower DNA load (7.03 ± 1.72 vs 7.82 ± 1.73; P < 0.001). In conclusion, our results observed vaccine-induced immune selection pressure in vaccine failed infants, substitutions in “a” determinant, especially the G145 substitution was still the most stable and remarkable site of vaccine escape.     

Prevalence of hepatitis B virus genotypes in chronic carriers in Santiago,Chile

The eight genotypes (designated A–H) of hepatitis B virus (HBV) display distinctive geographical distribution worldwide, with genotypes A, D and F frequently detected in South America. To determine the prevalence of HBV genotypes in Santiago, Chile, 131 samples from chronic carriers were used for PCR amplification, and genotyping was performed by RFLP. The results indicated that genotype F was the most prevalent among HBV carries (84% of the cases), whereas genotypes A, B, C and D were found at a prevalence of 3.8, 3.8, 6.1, and 2.3%, respectively. We discuss these data in the complex scenario of HBV epidemiology.     

Transmission routes of hepatitis B virus infection in chronic hepatitis B patients in The Netherlands

The Netherlands is a low endemic country for hepatitis B virus (HBV). Rotterdam, a city in The Netherlands harbors a large group of chronic hepatitis B (CHB) patients of which most are born abroad. The study included 464 consecutive CHB patients who were reported to the Municipal Public Health Service in Rotterdam from January 1, 2002 to September 15, 2005. The HBV genotypes, possible transmission routes of infection and travel history of CHB patients born in The Netherlands, were compared with those CHB patients living in The Netherlands but who were foreign-born, taking into account the ethnicity of the mother. Of the 464 patients with CHB infection, 14% were Dutch-born and 86% were foreign-born. The CHB patients in the Dutch-born group had genotypes A (35%), B (15%), C (11%), D (37%), and G (2%). In the foreign-born group, the distribution of genotypes was A (20%), B (15%), C (11%), D (40%), and E (15%). In the Dutch-born group, sexual transmission accounted for a larger proportion of infections (P < 0.0001) compared to the foreign-born group, whereas perinatal transmission is reported to be higher in the foreign-born group and in the Dutch-born group with a foreign mother. The genotypes of the chronic HBV strains determined corresponded well with the HBV genotypes expected from the countries of origin of the patients or their mothers. Genotypes A and D are predominant in CHB patients in The Netherlands.     

Limitations and improvements of the quantiplex branched-DNA assay in Hepatitis B virus-infected patients receiving lamivudine.

The branched DNA (bDNA) assay for hepatitis B virus (Chiron Corporation Emerville, USA) was investigated by application to HBV-infected patients in Taiwan, where the B and C genotypes of hepatitis B virus are most prevalent. The study group included sera with hepatitis B surface antigen (HBsAg) and e antigen (HBeAg); Group 1 (n=70) without treatment; Group 2 (n=28) lamivudine treatment less than 3 months; Group 3 (n=73) lamivudine treatment 3-12 months; Group 4 (n=45) HBeAg-negative sera after 1 year treatment with lamivudine; control group (n=36) HBsAg-negative sera. Comparison of identical-sample results showed a significantly higher coefficient of variation for low-level HBV DNA (<100 MEq/ml) than for high-level (> or =100 MEq/ml), indicating increasing assay inaccuracy uncertainty as the sample HBV DNA concentration decreased. It is thus concluded that low-titered sera should receive special careful pipetting and processing. It was also found that using the relative luminescence of the negative control plus two standard deviations (S.D.) as a new cutoff could promote sensitivity (97.1-->97.1%, 89.3-->100%, 76.7-->84.9%, and 17.8-->22.2% in Groups 1-4, respectively) and specificity (94.4-->97.2%). In summary, the bDNA HBV assay showed only moderate assay performance for samples with low HBV DNA levels. This problem can be improved partially by choosing a new cutoff value based on the relative luminescence of the negative controls in the kit.