Cost-effectiveness of additional hepatitis B virus nucleic acid testing of individual donations or minipools of six donations in the Netherlands

BACKGROUND: To further reduce the risk of hepatitis B virus (HBV) transmission by blood transfusion, nucleic acid testing (NAT) can be employed. The aim of this study is to estimate the incremental cost-effectiveness ratio (ICER) in the Netherlands of employing a triplex NAT assay aimed at HBV nucleic acid detection in individual donations (ID-NAT) or in minipools of 6 donations (MP-6-NAT), compared to a triplex NAT assay in minipools of 24 donations (MP-24-NAT).
STUDY DESIGN AND METHODS: A mathematical model was made of the whole transfusion chain from donors to recipients of blood in the Netherlands. The annual number of avoided HBV transmissions was estimated with the window-period incidence model. The natural history of a HBV infection in recipients is described by a Markov model.
RESULTS: The ICER of adding HBV MP-6-NAT or HBV ID-NAT in the Netherlands is €303,218 (95% confidence interval [CI], €233,001-€408,388) and €518,995 (95% CI, €399,359-€699,120) per quality-adjusted life-year, respectively. The ICER strongly correlates with the age of transfusion recipients.
CONCLUSION: The cost-effectiveness of additional HBV NAT is limited by the limited loss of life caused by HBV transmission. Despite a higher effectiveness, HBV ID-NAT is less cost-effective than MP-6-NAT due to higher costs. A future equivalent participation of immigrants from HBV-endemic countries in the donor base renders HBV NAT only slightly more cost-effective.     

拉米夫定治疗失代偿性乙型肝炎肝硬化的临床观察

目的:探讨拉米夫定治疗失代偿性乙型肝炎肝硬化的临床疗效及安全性。方法:64例失代偿性乙型肝炎肝硬化患者采用拉米夫定治疗,必要时给予利尿、人血浆白蛋白等对症支持治疗,并观察64例患者治疗前及治疗3、6、12个月后临床症状及体征和生化指标、血液学指标、病毒学指标改变及耐药发生情况。结果:与治疗前比较,64例患者治疗后,临床症状及体征明显改善(P<0.01);血清ALT、AST及总胆红素水平均下降(P均<0.01),血清白蛋白含量增加(P<0.05,P<0.01),Child-pugh评分下降(P<0.05,P<0.01);外周血白细胞及血小板计数水平均有明显增高(P均<0.01);血清HBV DNA定量水平显著下降(P<0.01);病毒耐药者12例,耐药发生率18.75%。结论:失代偿性乙型肝炎肝硬化患者长程拉米夫定治疗可有效阻止病情进展,并可提高患者生活质量。     

Performance characteristics of the COBAS AmpliScreen HIV-1 test, version 1.5, an assay designed for screening plasma mini-pools

BACKGROUND: The COBAS AmpliScreen HIV-1 test, version 1.5 (v1.5) (Roche Molecular Systems), is designed for screening pools composed of samples from 24 individual units of blood or plasma. A specimen-processing procedure (Multiprep) simultaneously concentrates and extracts HIV-1, HCV, and HBV particles from plasma and incorporates an HIV-1 internal control (IC) RNA. Processed samples are amplified by RT-PCR using HIV-1-specific primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes. STUDY DESIGN AND METHODS: Plasma samples containing known quantities of HIV-1 were used to evaluate analytical sensitivity and precision and to validate a pool testing algorithm. Analytical specificity was evaluated by adding various viruses and bacteria to HIV-1-negative plasma. Seroconversion panels were tested to estimate the window-period reduction achieved by RNA testing. RESULTS: The analytical sensitivity of the test (concentration that yields > or = 95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma. Representative strains from all HIV-1 group M subtypes were reproducibly detected (> 95% positive results) at concentrations of 20 to 200 viral particles per mL. The test did not cross-react with a set of 31 viral and 5 bacterial isolates, and it yielded negative results on a panel of 500 blood samples from HIV-1-seronegative donors. Plasma samples containing abnormally high levels of Hb, albumin, triglycerides, or bilirubin did not interfere with the test. HIV-1 RNA was detected 2 to 14 days before HIV-1 antibody and 0 to 28 days before p24 antigen. The test specifically detected pools containing a single positive unit with 2400 HIV-1 RNA copies per mL and correctly identified the positive unit. CONCLUSION: The COBAS AmpliScreen HIV-1 test, v1.5, has sufficient sensitivity to detect a single infected unit containing 600 copies of HIV-1 per mL in a pool with 23 uninfected units and should reduce the window period between infection and seroconversion by at least 2 to 14 days.     

COBAS Taqman HBVDNA的方法学验证

目的 评估COBAS Taqman HBV DNA分析系统的性能及临床应用。方法依据美国国家临床实验室标准化委员会（NCCLS）制订的临床化学方法评价方案（evaluation protocols），通过精密度、携带率、线性评价、仪器间比对、参考范围确认等试验，对COBAS Taqman HBV DNA分析系统的性能参数进行了评估。结果COBAS Taqman分析系统批内变异6．9％～8．6％，批间变异10．5％～18．7％，总变异10．8％～20．2％，线性范围6．0～10^7IU／ml，回收率98．7％～104．5％。仪器间比对相关性较好（r^2=0．998）。结论COBAS Taqman HBV DNA分析系统性能指标符合要求，操作简便、快捷，动力学范围广，是目前理想的HBV DNA检测方法。通过此次验证，建立了一套简便的核酸检测方法学评价方案，有利于临床实验室标准化操作的实施。     

COBAS Taqman HBV DNA的方法学验证

目的 评估COBAS Taqman HBV DNA分析系统的性能及临床应用.方法 依据美国国家临床实验室标准化委更会(NCCLS)制订的临床化学方法评价方案(evaluation protocols),通过精密度、携带率、线性评价、仪器同比对、参考范围确认等试验,对COBAS Taqman HBVDNA分析系统的性能参数进行了评估.结果 COBAS Taqman分析系统批内变异6.9%～8.6%,批间变异10.5%～18.7%,总变异10.8%～20.2%,线性范围6.0～101 IU/ml,回收率98.7%～104.5%.仪器间比对相关性较好(r2=0.998).结论 COBAS Taqman HBV DNA分析系统性能指标符合要求,操作简便、快捷,动力学范围广,是目前理想的HBV DNA检测方法.通过此次验证,建立了一套简便的核酸检测方法学评价方案,有利于临床实验室标准化操作的实施.     

Efficacy of hepatitis B virus (HBV) DNA screening and characterization of acute and occult HBV infections among blood donors from Madrid, Spain

BACKGROUND: Screening of blood units for hepatitis B virus (HBV) DNA identifies donations collected during the window period (WP) of the acute infection and may improve viral safety of the blood supply. It also leads to the detection of occult hepatitis B infection (OBI).
STUDY DESIGN AND METHODS: From January 2005 to December 2006, a total of 383,267 blood units were screened for hepatitis B surface antigen (HBsAg) and HBV DNA in two transfusion centers in Madrid, using either individual-donation nucleic acid testing (ID-NAT) or minipool (MP-NAT) of eight donations (MP8). Samples positive for HBV DNA and negative for HBsAg were confirmed by a second molecular test, the viral DNA was quantified, and a genome fragment including the region encoding the major hydrophilic region (MHR) of HBsAg was sequenced.
RESULTS: The overall yield of HBV DNA–positive, HBsAg-negative units was 1 in 21,282 (18 cases), higher when using ID-NAT than MP8-NAT (1:9862 vs. 1:51,011; p < 0.01). Four donations (1/95,817) were collected during the infectious pre-HBsAg WP, one during an early recovery stage, and the remaining 13 (1/29,482) were OBIs, six of whom had no detectable antibody to HBsAg. Low-level Genotype D HBV DNA was detected in all OBI cases; the frequencies of this genotype and MHR amino acid substitutions were significantly higher than reported from unselected Spanish HBsAg carriers. Donors with OBI had normal aminotransferase levels and were significantly older than donors carrying HBsAg.
CONCLUSIONS: Blood donors in the WP and with OBI are not uncommon in Madrid and are detected at a higher frequency with ID-NAT than MP-NAT.     

HBV BCP变异、前C区变异与HBeAg、HBV DNA的关系

目的 探讨慢性乙型肝炎HBV BCP变异、前c区变异与HBeAg表达、HBV DNA的关系.方法 HBV基因变异检测采用乙型肝炎病毒基因多态性检测芯片技术;血清HBV DNA水平和HBV-M的检测分别采用荧光定量核酸技术和ELISA法.结果HBV BCP变异株与HBeAg的阴转无显著相关性(P>0.05),但e抗原(+)/e抗体(-)组HBV DNA水平明显高于e抗原(-)/e抗体(+)组(P<0.001);HBV BCP联合前C区变异株与HBeAg阴性表达显著相关(P<0.001);且HBVDNA水平低于单纯HBV BCP变异组(P<0.05).结论 HBV BCP变异不能显著影响HBeAg的阴转,但可增加HBV DNA合成,使病毒复制进一步增强;前C区变异对HBeAg阴性的影响较大,但可导致HBV DNA的复制下降.     

Hepatitis B virus (HBV) in the elderly: an underestimated problem?

Hepatitis B infection in the aged can be underestimated as the clinical and serological pictures can be rather peculiar in these subjects. Institutions for the elderly carry an increased risk of HBV spread as do many other closed communities. People from lower socioeconomic class and from countries with a high HBV prevalence are less susceptible to infection as they are already immunized by previous infections. However, epidemics have been described in homes for the aged, mostly from those for wealthy people or from those in low prevalence countries. When infected the elderly tend to develop a subclinical hepatitis detected only by serum analysis. These infections are frequently followed by asymptomatic chronic carriage of HBsAg. This phenomenon may be due to alterations of the immune system in the aged, which is also suggested by the finding of very poor antibody responses to hepatitis B vaccines in the aged. Overt acute hepatitis is infrequent. It usually have a benign course, even if occasionally cholestatic. Very active type B chronic hepatitis is very rare, while most elderly patients with HBsAg-positive chronic liver disease have cirrhosis as the end stage of chronic hepatitis acquired previously.     

献血者血液乙型肝炎病毒表面抗原确认试验

目的 对使用国产HBsAg酶联免疫吸附试验(ELISA)一步法试剂盒检测无偿献血者血液(包括机采血小板献血体检者)的可靠性进行评价.方法 对无偿献血者血液样品,使用国产的HBsAg酶联免疫吸附试验(ELISA)试剂盒进行测定.结果 呈阳性反应者、3种试剂检测结果不一致的部分可疑结果者和3种试剂检测为阴性者,使用珠海丽珠试剂公司研发的乙型肝炎病毒表面抗原(HBsAg)确认试剂盒(中和试验法) 初步进行确认试验.确认试验方法参照丽珠公司提供的说明书进行.结果 132例样品中阳性反应或可疑阳性反应者123例被确认为阳性者106例,其中有17例可疑结果者常规确认试验结果为"阴性".经3种试剂再测,结果仍为可疑.9例0.074≤样品A值＜Cut off的样品和随机抽取的9例阴性样品常规确认试验结果仍为阴性.8例单一试剂呈阳性反应者常规确认为假阳性.结论 国产HBsAg ELISA试剂测定结果为阳性、弱阳性和可疑的样品应使用适当方法进行确认,排除假阳性,以保证结果准确.目前国产HBsAg酶联免疫吸附试验(ELISA)一步法试剂盒有较高的灵敏度,特异性尚待提高.     

Sensitivity of hepatitis B virus DNA transcription-mediated amplification testing in hepatitis B surface antigen-positive blood donations

BACKGROUND: The objective was to evaluate the performance of nucleic acid testing (NAT) in the detection of hepatitis B virus (HBV) infection in hepatitis B surface antigen (HBsAg)-positive blood donations. STUDY DESIGN AND METHODS: A total of 253 HBsAg- and anti-hepatitis B core antigen (HBc)-positive samples (50 hepatitis B e antigen [HBeAg]-positive and 203 anti-HBe-positive) from blood donations collected in France were studied. The samples were investigated with a blood screening assay (Procleix Ultrio, Chiron/Gen-Probe) in minipool (MP; x8) and in individual-donation (ID) testing. All nonreactive samples were retested once, and nonreactive MP samples were assayed for viral load (VL). RESULTS: All 50 HBeAg-positive samples were reactive in MP-NAT and ID-NAT. Of the 203 anti-HBe-positive donations, 80.3 percent were MP- and ID-reactive, 17.2 percent were MP-nonreactive and ID-reactive, and 2.5 percent were nonreactive in ID-NAT. Overall the sensitivity of ID-NAT was 98 percent versus 84 percent for MP-NAT. After retesting, 16 of the 35 MP-nonreactive and/or ID-reactive donations became MP-reactive and 2 of the ID-nonreactive donations became NAT-reactive. The capacity of Procleix Ultrio to detect HBV DNA was not related to HBsAg subtype, but correlated with the VL: the mean VL in the group of MP-nonreactive samples was 1,420 copies per mL vs. 17,000 copies per mL in the group of 40 MP-reactive samples. CONCLUSION: These results demonstrate that HBV-NAT in ID format is far more effective in detecting viremia in chronic HBsAg carriers than in MP-NAT. The sensitivity of the NAT assay needs to be improved to be considered for replacing the current HBsAg assays, especially when anti-HBc testing is not performed.     

血清中游离乙型肝炎病毒DNA检测分析

目的了解乙型肝炎患者血清中游离乙型肝炎病毒DNA(HBVDNA)的情况。方法血清加DNA提取液处理后检测的DNA作为总的HBVDNA,血清加蒸馏水直接检测的DNA作为游离的HBVDNA,用实时荧光定量PCR法检测DNA,用生化自动分析仪测定标本的ALT和AST。结果30例标本中,有27例标本检出有游离HBV DNA,检出率为70.0%,总HBVDNA在105数量级以上(包括105数量级),均检出有游离HBV DNA。总HBV DNA平均载量为(1.6±3.1)×108copies/ml,游离HBVDNA平均载量为(4.8±8.2)×106copies/ml,游离HBV DNA与总HBV DNA含量呈正相关关系(r=0.545,P0.05)。在30例中,有20例肝功能正常,有10例肝功能不正常。在肝功能正常组中有14例检出有游离HBVDNA,检出率为70.0%(14/20);在肝功能不正常组中有7例检出有游离HBV DNA,检出率为70.0%(7/10);两组检出的游离HBV DNA平均含量比较无统计差异(P0.05)。结论在乙型肝炎患者血清中,当总HBVDNA在105数量级以上(包括105数量级),血清中就含有游离HBV DNA,与总HBV DNA含量呈正相关关系;游离的HBVDNA的释放与肝细胞是否受损没有关系。     

Correlation of improved hepatitis B surface antigen detection limits with hepatitis B virus DNA nucleic acid test yield in blood donations

BACKGROUND: This study provides data on the quantitative relationship between hepatitis B surface antigen (HBsAg; ng/mL) and hepatitis B virus (HBV) nucleic acid test (NAT; copies/mL) and addresses whether HBsAg assays with improved sensitivity would impact the detection of HBV‐positive samples from occult or early seroconversion window period infections. STUDY DESIGN AND METHODS: Plasma samples were tested with an HBsAg assay (PRISM, Abbott Laboratories; sensitivity, 0.08‐0.1 ng/mL) and with two HBsAg research prototype assays: HBsAg Prototype 1 (sensitivity, 0.032‐0.045 ng/mL) and HBsAg Prototype 2 (sensitivity, 0.009‐0.017 ng/mL); NAT assays were used to determine HBV DNA copy levels. RESULTS: Samples from 10 hepatitis B seroconversion panels covering the ramp‐up phase were utilized to examine the relationship between detection of HBsAg using improved assays and viral load using quantitative HBV DNA polymerase chain reaction. For these samples, detection at the HBsAg assay cutoff (sample‐to‐cutoff ratio, 1.0) corresponded to 206 copies/mL HBV DNA for the HBsAg Prototype 1 assay and 329 copies/mL for the PRISM HBsAg assay. Compared to the PRISM HBsAg and HBsAg Prototype 1 assays, the HBsAg Prototype 2 assay detected two additional samples of 32 HBV DNA–positive samples obtained from blood donors with occult HBV and one of seven from blood donors with early window period infections. CONCLUSION: Increased sensitivity HBsAg assays result in the detection of samples containing lower viral loads. Improvements in the analytic sensitivity of HBsAg prototype assays allow the detection of additional HBV DNA–positive samples from donors with window period or occult infections compared to PRISM HBsAg. Improved HBsAg assays should allow for incremental detection of HBV infection.     

探讨乙型肝炎病毒前S1抗原和乙型肝炎病毒DNA的相关性及临床意义

目的探讨乙型肝炎（乙肝）病毒（HBV）前S1（PreS1）抗原和HBV-DNA的相关性及其在乙肝诊断中的临床意义。方法采用酶联免疫吸附试验（ELISA）法对290例乙肝血清进行PreS1和HbsAg、HbsAb、HbeAg、HbeAb、HBcAb检测,同时对每个标本进行HBV-DNA定量分析。根据乙肝标志物的检测结果,将所测标本中HBsAg、HBeAg、HBcAb阳性90例设为A组;HBsAg、HBeAb、HBcAb阳性95例设为B组;HBsAg、HBcAb阳性65例设为C组;HBsAg阳性40例设为D组。比较各组PreS1和HBV-DNA的阳性率。结果 290例检测标本中,PreS1抗原阳性183例,阳性率63.1%,其中A组74例（82.2%）,B、C、D组分别为52例（54.7%）、45例（69.2%）、12例（30.0%）,HBeAg阳性者（A组）PreS1抗原阳性率82.2%显著高于HBeAg阴性者（B、C、D组）54.5%（P〈0.01）。HBV-DNA阳性167例,阳性率为57.6%,其中A组85例（94.4%）,B、C、D组分别为42例（44.2%）、35例（53.8%）、5例（12.5%）,HBeAg阳性者（A组）HBV-DNA阳性率94.4%显著高于HBeAg阴性者（B、C、D组）41.0%（P〈0.01）。HBV-DNA阳性患者中,HBeAg阳性85例中51例（60.0%）PreS1抗原阳性,HBeAg阴性的82例中50例（61.0%）PreS1抗原阳性,两组比较差异无统计学意义（P〉0.05）。结论血清乙肝病毒PreS1和HBV-DNA有很高的符合率,且较HBeAg更能准确反映HBV复制的情况,可同时作为监测HBV感染复制及预后的指标。     

乙型肝炎感染者不同血清标志物HBV DNA的表达及意义

目的探讨血清HBV DNA水平与HBV标志物（HBVM）表现模式的相关性。方法血清HBV DNA定量榆测采用PCR ELISA法，HBVM采用ELISA法。结果在HBVM阳性的430例血清中，有232例HBVDNA阳性，占54，0％。血清HBsAg、HBeAg、抗-HBc阳性者HBVDNA阳性率占94．8％，≥106copies／ml占65．9％；HBsAg、抗-HBe、抗-HBc阳性者HBV DNA阳性占29．4％，≥106copies／ml占17，5％。结论血清HBVM阳性患者中约54．0％的血清HBV DNA阳性，且HBeAg阳性患者中HBV DNA含量及阳性率明显高于抗-HBe阳性者；在抗-HBs、抗-HBc阳性或HBsAg阴性者血清内也有HBVDNA阳性者。     

血液HBV DNA全自动检测及基因分型分析

目的建立血液HBVDNA标本汇集、核酸提取、扩增及检测的全自动筛查模式,对阳性标本进行基因分型和血清学追踪检测,为血液HBVDNA自动化筛查提供科学依据。方法在酶联免疫吸附试验(ELISA)筛查血液基础上,应用STAR2000加样仪自编程序进行全自动血样汇集(24人份),在MPLC仪上全自动提取标本核酸,应用PCR方法在COBASAMPLICOR进行扩增和检测分析结果,用国际标准核酸质控品考评检出限量,对阳性标本进行基因分型并追踪血清转换过程。结果全自动汇集、全自动核酸提取和扩增及检测HBVDNA95%检出限量为38.9IU/ml,95%CI为(21323)。通过对16512个标本共688个汇集池分析,HBVDNA阳性8例,阳性率为0.049%。其中C型3人,B型2人,D型1人,2人未能确定基因型,6例阳性标本追踪发现,3例发生了血清转换现象。结论HBVDNA全自动核酸检测方法可应用于24人份血液混样核酸筛查。     

Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection.

It has been suggested that immune selection pressure exerted by the cytotoxic T lymphocyte (CTL) response could be responsible for viral persistence during chronic hepatitis B virus infection. To address this question, in the current study we compared the DNA and amino acid sequences of, and the CTL responses to, multiple HLA-A2-restricted CTL epitopes in the hepatitis B virus in several HLA-A2-positive patients with acute and chronic hepatitis. Our results indicate that the CTL response to these epitopes is barely detectable in the majority of patients with chronic hepatitis. Further, we show that the weak CTL response is not secondary in infection by mutant viruses lacking these epitopes, and we show that the CTL response did not select for escape mutants in any of these patients. We conclude that an ineffective hepatitis B virus specific CTL response is the primary determinant of viral persistence in chronic hepatitis and that immune selection of viral variants is not a common event in the majority of patients.     

Multicenter evaluation of a commercial multiplex polymerase chain reaction test for screening plasma donations for parvovirus B19 DNA and hepatitis A virus RNA

BACKGROUND: Three European laboratories evaluated the TaqScreen DPX test (DPX test), a multiplex nucleic acid test assay for the simultaneous detection and quantitation of parvovirus B19 (B19V) DNA and the detection of hepatitis A virus (HAV) RNA. STUDY DESIGN AND METHODS: The 95% limit of detection of the test for B19V and HAV was determined using the respective WHO International Standards. The reproducibility of the test was evaluated by testing replicate samples of B19V at log 4.0 and 40 IU/mL and HAV at 5 IU/mL. The accuracy of the DPX test for B19V was evaluated by replicate testing of B19V samples containing log 3.0, log 4.0, and log 5.0 IU/mL. Panels of B19V Genotypes 1, 2, and 3 and HAV genotypes were evaluated. Cross‐contamination was evaluated. For comparison of the DPX test and the established tests, the sites tested plasma samples in pools of either 96 or 480 donations. RESULTS: The mean 95% lower limits of detection of the three laboratories for B19V and HAV were 20.30 and 1.85 IU/mL. The test showed good reproducibility with the major part of the variance of the test being attributed to intermediate assay variation. The test showed great accuracy for B19V, especially at log 4.0 IU/mL. Spiking of test pools of 480 donations and manufacturing pools with log 4.0 IU/mL B19 DNA and 4 IU/mL HAV RNA showed that the DPX assay was robust. The test was able to detect the three genotypes of B19V and HAV genotypes. No cross‐contamination was seen. Test results of routine samples correlated well with those of the established tests. CONCLUSION: The DPX test is a robust and sensitive test for the detection of B19V and HAV in plasma samples. The quantitative B19V results obtained with the test are accurate, and the test is able to detect all the known genotypes of B19V and HAV and fulfills all the European Pharmacopoeia and Food and Drug Administration requirements for a B19V and HAV test for screening of plasma donations and samples from plasma pools for manufacture.