合格献血者血浆HBV残留情况分析

目的调查合格献血者血浆残留HBV的阳性率,评估输血后HBV感染风险。方法收集945份合格献血者的血浆,采用ELISA法检测HBV核心抗体（抗-HBc）;提取血浆DNA,采用巢式PCR方法检测HBV DNA。结果血浆标本以1∶5稀释后抗-HBc阳性率为19.68%,而1∶50稀释后阳性率则为2.33%。共检测出HBV DNA阳性标本3例,其中1例抗-HBc阳性,另外2例抗-HBc为阴性。合格献血者中隐匿性HBV感染率约为0.32%。结论我国合格献血者标本中仍有部分血浆有HBV残留,抗-HBc阳性标本中HBV DNA阳性率较抗-HBc阴性标本高,应引起重视。     

Hepatitis B virus DNA in blood donors with anti-HBc as a possible indicator of active hepatitis B virus infection in Yucatan, Mexico

Hepatitis B virus (HBV) may be present in serum even when negative for HBV surface antigen (HBsAg). If routine screening of sera for anti-HBV core antigen (anti-HBc) is not done, low-level HBV viraemia may not be identified. A study was done on the presence of HBV DNA in serum samples from Mexican blood donors negative for HBsAg. Sera from 158 volunteer blood donors, negative for HBsAg and anti-HBs, but positive for anti-HBc, were analysed using nested polymerase chain reaction (PCR). HBV DNA was detected in sera from 13 (8.23%) of the 158. Specificity of the PCR-amplified products was corroborated using Southern blot. Single strand conformation polymorphism (SSCP) analysis showed identical SSCP-banding patterns for all 13 PCR products, suggesting similar cDNA sequences. Occult HBV infection was observed in approximately 8% of anti-HBc only donors. The absence of HBsAg in the blood of apparently healthy individuals may not be sufficient to ensure lack of circulating HBV, and blood containing anti-HBc only may be infectious until proven otherwise.     

青岛地区无偿献血者乙型肝炎病毒感染血清学和病毒特征分析

目的 分析青岛地区无偿献血者乙型肝炎病毒感染的血清学及病毒学特征.方法 采用常规的血清学试验和核酸扩增技术对本地区315 520份无偿献血者标本进行联合筛查,对HBsAg-/HBV DNA+标本进行高精度病毒载量检测和补充乙肝5项检测,采用PCR直接测序法获得标本中HBsAg编码基因即S基因序列,分析病毒基因型别及氨基...     

Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay

BACKGROUND: Studies were conducted using samples from early and late-stage HBV-infected persons to determine the pool size at which PCR had better sensitivity than a sensitive HBsAg chemoluminescence immunoassay (CLIA-HBsAg). STUDY DESIGN AND METHODS: HBV seroconversion panels were tested for HBsAg by CLIA and for HBV DNA by nested PCR (95% hit rate: 100 copies/mL); PCR was carried out at various dilutions. HBV serologically positive samples that were detected from the simultaneous screening of 540,161 routine whole-blood donations using CLIA-HBsAg and agglutination assays were also characterized for additional markers of HBV infection. RESULTS: In 9 of 10 HBV seroconversion panels, PCR had better sensitivity than CLIA-HBsAg at dilutions of 1-in-25 or lower. Of 65 CLIA-only confirmed-positive donor samples (agglutination assay-negative), 8 represented early infection, 2 of which were PCR positive at a 1-in-50 dilution but negative at a 1-in-100 dilution. Only 2 of 47 samples from probable late-stage HBV infection that were positive on CLIA only were PCR positive with 0.1-mL sample volume and the S-region primer; the remaining 45 samples required a 1.0-mL sample input and C-region primer for increased PCR positivity. The remaining 10 CLIA-only confirmed-positive donor samples were from HBV vaccine recipients. None of the 12 CLIA- and HBsAg-negative donor samples that were strongly anti-HBc reactive could be detected by PCR at any dilution; all 12 were PCR positive when undiluted, but 4 required a 1.0-mL input volume for PCR positivity. CONCLUSION: For the detection of samples representing early-stage HBV infection, PCR at dilutions of 1-in-25 or lower (equivalent to a pool of < or =25 members) had greater sensitivity than CLIA-HBsAg. In contrast, samples from late-stage HBV infection were detected by PCR only with undiluted samples (0.1-mL or 1.0-mL input volumes), regardless of CLIA-HBsAg reactivity. Therefore, although NAT using minipools of 25 donations or less may be effective for the detection of early-stage HBV infection, it may not be effective for the detection of persistent HBV infection.     

Relapses and reinfections in viral hepatitis: an analysis of repeated admissions

The causes of repeated admissions of 35 patients with viral hepatitides were unraveled on the basis of studies of the serum markers HAV, HBV, HBsAg, anti-HBs, HBeAg, IgM-anti-HBc, anti-HBc, IgM-anti-HAV, anti-HAV, and anti-D. Mixed viral infections were diagnosed in 14 persons: 3 persons had superinfection with HAV associated with chronic HBsAg carriership, 6 had superinfection with HDV and 5 manifested superinfection with HAHB viruses. Reactivation of chronic hepatitis was diagnosed in 5 patients. The measurement of the activity of alanine aminotransferase in blood serum diluted 1:10 and of IgM-anti-HBc titers was shown to be of high differential diagnostic importance in the identification of relapses and reinfections. The effects of superinfections with HAV and HDV are under discussion.     

Infectivity of blood from PCR-positive, HBsAg-negative, anti-HBs-positive cases of resolved hepatitis B infection

BACKGROUND: Numerous reports have noted the existence of sera, particularly from resolving cases of HBV infection, that are positive for HBV DNA by PCR, despite being negative for HBsAg and IgM anti-HBc. If such blood is infective and detectable by HBV NAT screening, it seems desirable to introduce such screening for transfused blood. STUDY DESIGN AND METHODS: Three chimpanzees were inoculated with serum and lymphocytes from three patients who were HBV DNA PCR positive, but HBsAg negative. The animals were tested over a period of 15 months for HBsAg, anti-HBs, anti-HBc, and HBV DNA by PCR. RESULTS: All animals remained uninfected. CONCLUSION: Small amounts of plasma and MNCs from HBV DNA-positive HBsAg-negative blood do not appear to be infectious; however, further studies with larger volumes of inoculum should be conducted.     

Liver transplantation-associated de novo hepatitis B virus infection: application of molecular evolutionary analysis

OBJECTIVE: De novo hepatitis B virus (HBV) infection after liver transplantation has recently been reported to be associated with donors without serum hepatitis B surface antigen (HbsAg) but with hepatitis B core antigen (anti-HBc). We elucidate the source of de novo HBV infection after liver transplantation by molecular evolutionary analysis. METHODS: The serum sample was obtained from a recipient who underwent living related liver transplantation. He was negative for all HBV-related serum markers before the transplantation. The recipient became seropositive for HBsAg at 6 months after transplantation. The liver tissue was obtained from a donor who was seronegative for HBsAg, but positive for anti-HBs and anti-HBc. RESULTS: HBV DNA was detected from the serum and liver tissue in a recipient and donor, respectively. A total of 5 clones each of small-S gene of HBV from the donor and recipient were sequenced. A phylogenetic tree analysis based on small-S gene revealed that all isolates derived from the recipient and donor were clustered together within a close range of evolutionary distances. These results indicated that HBV was transmitted by the liver graft from the donor. CONCLUSIONS: Molecular evolutionary analysis can be adopted for the study of the transmission route of viral infection via organ transplantation.     

Evaluation of serologic screening of blood donors in India reveals a lack of correlation between anti-HBc titer and PCR-amplified HBV DNA

BACKGROUND: Transfusion associated-HBV (TAHBV) is estimated at approximately 1.5 percent in postsurgical recipients and 50 percent or more in multiple-transfusion recipients in India. Not transfusing blood with high-titer anti-HBc, which reportedly correlates with the presence of HBV DNA, helped reduce TAHBV in Japan. This study tested anti-HBc-reactive donors for PCR-amplified HBV DNA and its correlation with anti-HBc titers. STUDY DESIGN AND METHODS: In total, 30,853 donors from Cohort 1 (24,694 in 2001) and Cohort 2 (6159 in 2000) were screened for anti-HBc and anti-HBs. Amplification of HBV DNA was performed on an unselected subset of 147 out of 3304 anti-HBc-only sera from Cohort 1 and 230 out of 6159 from Cohort 2. Correlation of anti-HBc titers in DNA positive (n = 48), DNA negative (n = 40), anti-HBs reactive (n = 44), and HBsAg reactive (n = 45) donors was by Fisher's exact test. RESULTS: In Cohort 1, 2673 (10.82%) donors were reactive for anti-HBc, of whom 1038 (4.20%) were anti-HBc only. HBV DNA was detected in 40 out of 147 (27.21%) and 48 out of 230 (20.87%) donors with anti-HBc only from the two cohorts. Anti-HBc titers detected no significant difference between the first three groups. CONCLUSION: Cryptic HBV infection was observed in approximately 25 percent of anti-HBc-only donors. No correlation was established between HBc titers and presence of HBV DNA.     

Absence of hepatitis B virus DNA detected by polymerase chain reaction in blood donors who are hepatitis B surface antigen negative and antibody to hepatitis B core antigen positive from a United States population with a low prevalence of hepatitis B serologic markers

A recent study in hepatitis B surface antigen (HBsAg)-negative, antibody to hepatitis B core antigen (anti-HBc)-positive blood donors from a population with a high prevalence of hepatitis B serologic markers showed the presence of hepatitis B virus DNA (HBV DNA) as detected by polymerase chain reaction (PCR) in 4 percent of these donors. A sensitive, nested PCR assay was used to assess the prevalence of HBV DNA in a population of HBsAg-negative, anti-HBc-positive blood donors from a United States population with a low prevalence of hepatitis B serologic markers. The lower limit for detection by the PCR assay was 10(-5) pg per mL of HBV DNA. There was a review of 26,492 consecutive blood donations in a 12-month period. During this time, only 1 unit (0.004%) was HBsAg positive. An additional 158 units (0.6%) were repeatably reactive for anti-HBc. These 158 HBsAg-negative, anti- HBc-positive units were given by 119 donors of blood for allogeneic and autologous use. HBV DNA was not detected by PCR in blood from 83 allogeneic blood donors (93 samples) or 36 autologous blood donors (65 samples). Anti-HBc testing is an inefficient means of screening for potential hepatitis B infectivity and is associated with low test specificity in populations with a low prevalence of hepatitis B serologic markers.     

Surface gene mutations of hepatitis B virus among high-risk patients with occult hepatitis B virus infection

Surface gene mutants of hepatitis B virus (HBV) have been reported in a variety of patient groups. Because of limited data regarding these mutations in patients with occult HBV infections; we aimed to determine these mutations among high-risk patients with occult HBV infection. The presence of HBV-DNA was determined in patients with isolated anti-HBc by real-time polymerase chain reaction (PCR). Then, surface gene region was amplified by nested PCR and mutations were analyzed after sequencing. The mutations that resulted in nonfunctional hepatitis B surface antigen (HBsAg) were insertion of single nucleotide in 2 cases, which causes frameshift and single-nucleotide replacement, and premature stop codons at Leu15 and Gly10 in the other 2 cases. Amino acid substitution at amino acid position 207(S207N) was found in the other isolates. Our study suggested that “a” region mutations did not play a major role in HBsAg detection, and other genetic and nongenetic factors may be responsible for failure to detect HBsAg by routine laboratory tests.     

上海地区献血人群隐匿性HBV感染状况

目的 了解HBsAg阴性、抗-HBc阳性的献血人群中HBV DNA感染情况.方法采用酶联免疫法(ELISA)检测5 121份HBsAg阴性的合格献血者血清抗-HBc和抗-HBc阳性反应滴度;对抗-HBc阳性样本采用ELISA法检测血清抗-HBs,采用巢式PCR三区段扩增法检测HBV DNA.结果HBsAg阴性的献血人群...     

Frequent presence of HBV in the sera of HBsAg-negative, anti-HBc-positive blood donors

BACKGROUND: Recent studies have revealed that HBV may not be cleared even after the disappearance of serum HBsAg. The purpose of this study was to investigate whether the replication of HBV persists in HBsAg-negative blood donors who lack apparent liver disease. STUDY DESIGN AND METHODS: Serum HBV was examined by using PCR coupled with Southern blotting in 50 blood donors who were identified to be HBsAg negative but anti-HBc positive. RESULTS: HBV DNA was detected in the sera from 19 (38%) of 50 donors. In 11 of the 19, HBV existed exclusively as immune complexes, while HBV presumably did not exist as immune complexes in the remaining eight. The levels of HBV DNA were similar to those in patients who had recovered from acute HBV. Some nucleotide substitutions, which did not confer amino acid changes in the major epitope of HBsAg, were found in the preS-S regions. CONCLUSION: The replication of HBV is ongoing in a substantial proportion of healthy blood donors who have anti-HBc. Blood from such donors may contain very low levels of HBV free of immune complex formation and should be excluded for transfusion. The fact that such blood donors apparently lacked liver disease suggests no pathogenicity of such "occult" HBV.     

乙型肝炎病毒核酸载量与血清学标志物相关性的研究

目的分析HBVDNA载量与血清学标志物的相关性。方法应用乙型肝炎病毒核酸试剂定量检测468份献血员样本HBVDNA含量,并分别定性或定量检测HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc及抗-HBcIgM血清学标志物,计算不同病毒核酸水平样本中5个乙肝血清学标志物阳性率及HBsAg、抗-HBs水平的分布情况。结果HBVDNA阴性样本中HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc阳性率分别为12.8%、39.8%、0、21.1%、49.6%,而HBVDNA阳性样本中HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc血清学标志物阳性率分别为91.3%、0.7%、20.9%、65.0%、88.6%,血清学指标在不同HBVDNA水平的阳性率差异具有统计学意义(χ2=197.4,P<0.01)。HBVDNA载量与HBsAg水平成正相关性,与抗-HBs水平成负相关性。结论低水平的HBsAg在不同HBVDNA载量水平的样本均有分布,HBsAg检测能力需要进一步提高,另外在我国人群中存在低乙型肝炎病毒核酸水平携带者,乙型肝炎病毒核酸可以弥补血清学检测试剂的不足。     

Transfusion-transmitted HBV infection in an endemic area: the necessity of more sensitive screening for HBV carriers

BACKGROUND: By NAT, HBV DNA is occasionally detectable in blood donors with past HBV infection but negative for HBsAg. Whether or not these donors can cause transfusion-transmitted HBV infections is uncertain. STUDY DESIGN AND METHODS: To determine whether or not donors with past HBV infection but negative for HbsAg can cause HBV transfusion-transmitted infections, recipients followed for blood transfusion in a university medical center in Taiwan were studied. HBV DNA and serologic markers were tested in donors and recipients. RESULTS: Of 1,038 enrolled recipients, 910 completed the 6-month post-transfusion follow-up visit. Of these, only 39 patients (4.3%) tested negative on the pretransfusion sample for HBsAg, anti-HBs, anti-HBc, and HBV DNA by PCR. These 39 HBV-naive recipients had been transfused with blood from 147 donations for which stored samples were available for HBV DNA testing by PCR; 11 of these HBsAg-negative samples tested positive for HBV DNA and anti-HBc. Two of the 11 patients who received the HBV-DNA-positive donations (18%) became positive for HBV DNA, and one seroconverted to anti-HBc and finally to anti-HBs, with a mild transient elevation of serum ALT activities. Based on the one confirmed case of HBV transmission, a projection was made that approximately 200 post-transfusion HBV infections could occur in one million units of transfused blood in Taiwan. CONCLUSIONS: In HBV-endemic areas like Taiwan, where blood donors are screened for HBsAg only, the risk of transfusion-transmitted HBV appears to be substantial. Implementation of NAT for blood screening in these settings warrants consideration.     

苏州地区抗-HBc阳性合格青年献血人群隐匿性HBV感染的血清学及分子生物学特性分析

目的探讨苏州地区乙型肝炎核心抗体(抗-HBc)阳性合格的青年献血人群隐匿性HBV感染的生物学及血清学特性。方法选取2013年10月至2016年6月乙型肝炎表面抗原(HBsAg)阴性、核酸检测(NAT)有反应性的青年献血者120例作为研究对象,进行乙型肝炎表面抗体(抗-HBs)定量检测和乙型肝炎两对半检测,对抗-HBc阳性标本进行病毒核酸提取和基本核心启动子区(BCP区)/前C区(PC区)及S区巢式PCR扩增,对扩增结果阳性标本进行基因测序及序列分析。结果 120例献血者中,抗-HBc(+)31例,其中22~25岁组25例,18~21岁组6例,差异有统计学意义(P0.05);抗-HBs(+)89例,其中22~25岁组16例,18~21岁组73例。利用巢式PCR扩增法对抗-HBc(+)标本进行PCR扩增,其中1例BCP区阳性,2例S区阳性,均属于22~25岁组。S区阳性标本的分型和测序结果显示:2例均为B型HBV,与野生型DNA序列相比,其中1例存在氨基酸序列E44U变异,1例存在T532G变异。结论对于HBsAg检测合格的抗-HBc(+)青年献血者,并不能保证其血液中一定不含有HBV DNA。为降低HBV经输血传播的风险,仍然需要进一步提高乙肝高流行地区核酸检测方法的灵敏度。     

Prevalence and occurrence of infections caused by hepatitis B virus in the dialysis units in the Apulia region

Sera of 803 hemodialysis patients and 413 staff members were tested to evaluate the relationship between infectivity markers and spread of HBV infection in dialysis units. HBsAg was detected in 13.8% patients undergoing chronic hemodialysis and 3.9% staff members. High prevalence of HBeAg and DNA polymerase activity was observed only in HBsAg positive patients. The highest titers of HBsAg and anti-HBc were detected in hemodialysis patients, whereas asymptomatic carriers showed low titers of these markers. A highly significant correlation was recorded between detection of HBeAg in patients and presence of serum DNA activity. These data suggest that in HBsAg hemodialysis patients a more active viral replication occurs and a higher contagiousness of these subjects.     

155例乙型肝炎血清学标志物少见模式的分析

目的对155例乙型肝炎血清学标志物(HBV-M)少见模式进行分析,探究可能原因。方法用酶联免疫吸附试验(ELISA)检测患者HBV-M,同时参考聚合酶链反应(PCR)HBV DNA检测的结果进行综合分析。结果发现155例少见HBV-M结果模式,主要表现为三组:HBsAg与抗-HBs同时阳性的HBV感染;抗-HBc阴性的HBV感染;HBsAg阴性的HBV感染。结论机体免疫反应的不同、ELISA检测方法本身的局限性、病毒变异都可能导致HBV-M检测结果出现少见模式,建议使用更为灵敏的化学发光法定量检测。     

Quantification of hepatitis B virus genomes and infectivity in human serum samples

BACKGROUND: Hepatitis B virus (HBV) infections are still a major health issue, with approximately 350 million people chronically infected with HBV worldwide. Information about the minimum copy number of HBV genomes required for infection would be useful as a reference for drug and vaccine development; for monitoring HBV patients during treatment; for screening of blood, organ, and tissue donors; and for regulating nucleic acid amplification assays for HBV. STUDY DESIGN AND METHODS: Serum samples from chronic carriers (hepatitis B surface antigen-positive and antibody to HBV core antigen-positive) of the three most common subtypes of HBV were studied; their infectivity titers had been evaluated previously in chimpanzees. The genotypes of the HBV samples were determined by DNA sequences and type-specific amino acids of the S gene of HBV. Copy numbers of HBV DNA were quantified by real-time TaqMan polymerase chain reaction (PCR) and by nested PCR applied to limiting dilutions. The copy number determined for each inoculum was compared with previously defined chimpanzee infectivity titers. RESULTS: The genotypes of the HBV adw, ayw, and adr inocula were A, D, and C, respectively. The concentration of HBV DNA was determined to be 5.4 x 10(9), 2.5 x 10(9), and 3.1 x 10(8) genome equivalents (geq) per mL for serum samples containing the adw, ayw, and adr, respectively. The chimpanzee infectivity titers per milliliter of these initial HBV-containing serum samples were previously determined to be 10(7.5) for adw, 10(7.5) for ayw (MS-2 strain), and 10(8) for adr. CONCLUSION: The minimal copy number of HBV DNA in chronic carriers of HBV that can infect the chimpanzee model was estimated to be from 3 to 169 geq based upon the three well-characterized inocula.     

人工肝和肝移植术后的乙肝三系统定量观察

目的观察人工肝技术和肝移植手术对重症乙肝患者乙肝病毒标志物的影响。方法乙肝三系统定量采用时间分辨免疫荧光技术,乙肝病毒DNA定量采用实时荧光定量PCR法。结果28例实施人工肝技术的重型肝炎患者治疗前后的乙肝三系统定量和HBVDNA定量结果为：HBsAg171．19&#177;32．10ng／mL,HBeAg0．03&#177;0．029NCU／mL,抗-HBe3．97&#177;4．61NCU／mL,抗-HBc5．98&#177;3．31NCU／mL,HBVDNA（1．1&#215;10^7）&#177;（6．81&#215;10^6）copy／mL和HBsAg168．14&#177;39．40ng／mL,HBeAg0．02&#177;0．023NCU／mL,抗-HBe3．95&#177;4．34NCU／mL,抗-HBc6．41&#177;3．13NCU／mL,HBVDNA（1．1&#215;10^7）&#177;（6．23&#215;10^6）copy／mL。P值均大于0．05,差异无统计学意义。26例施行肝移植手术的患者治疗前后的乙肝三系统定量和HBVDNA定量结果分别为HBsAg144．65&#177;77．00ng／mL,HBeAg0．02&#177;0．028NCU／mL,抗-HBe4．32&#177;6．43NCU／mL,抗-HBc6．04&#177;4．88NCU／mL,HBV DNA（1．0&#215;10^7）&#177;（6．89&#215;10^6）copy／mL和HBsAg6．54&#177;3．32ng／mL,HBeAg0．02&#177;0．016NCU／mL,抗-HBe4．79&#177;6．44NCU／mL,抗-HBc5．97&#177;4．64NCU／mL,HBV DNA（1．04&#215;10^2）&#177;（3．40&#215;10^2）copy／mL。除抗-HBe和抗-HBc,P值大于0．05外,其他项目P值均小于0．05,差异有统计学意义。结论肝移植手术可有效清除乙肝患者体内的乙肝病毒,人工肝支持系统虽不能有效清除乙肝患者体内的乙肝病毒,但可改善重型肝炎患者体内严重紊乱的内环境,为肝移植手术创造合适的条件和机会,赢得宝贵时间。     

液相杂交技术在聚合酶链反应酶联免疫检测的应用

目的采用液相杂交技术,简化核酸杂交过程,建立乙肝病毒（HBV）的液相杂交的聚合酶链反应-酶联免疫检测（PCR-ELISA）方法。方法将5′端标记生物素的PCR扩增产物与5′端标记地高辛的探针呈液相混合,90℃2min,55℃1min杂交。杂交后产物被链霉亲和素酶标板固定,经酶标记抗地高辛标记抗体结合、显色。结果液相杂交酶联免疫检测条件优化探针浓度3pmol/次,酶标记抗体稀释度1∶1000。液相杂交时间3min,固相杂交时间120min,但其检测结果无明显差别。检测灵敏度1×10