化学发光法和酶联免疫法作为筛选试验测定丙肝抗体的评价

目的比较化学发光法(CLIA)和酶联免疫法(EIA)测抗-HCV对诊断HCV感染的检出率,分析CLIA测定抗-HCV作为HCV感染初筛试验的重要临床意义。方法分别用CLIA、EIA方法检测所有临床标本中的抗-HCV,选取抗-HCV初筛阳性样本进行HCV RNA确认试验。结果 6461例样本中,EIA和CLIA测抗-HCV阳性率分别为2.86%和3.17%;确认试验中,CLIA法抗-HCV阳性与HCV RNA的符合率为97.1%,显著高于EIA组(91.9%)。EIA法测抗-HCV,S:CO>5.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为69.7%;CLIA法测抗-HCV,S:CO>2.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为92.9%。有31例样本CLIA法抗-HCV和HCV RNA阳性,而EIA法抗-HCV阴性;有4例样本EIA法检测为阳性,而CLIA法和PCR确认试验均为阴性。结论CLIA法测抗-HCV作为HCV感染初筛实验,与传统EIA相比,特异性和阳性预测值更高,有效降低了假阳性率,有利于临床医生对HCV感染患者的早期诊断和治疗。     

实时荧光定量HCV RNA与ALT检测在丙肝筛查中临床价值的研究

目的 探讨抗-HCV抗体阳性时,实时荧光定量HCV RNA与ALT检测在丙肝筛查中临床价值的研究.方法 回顾选择2019年1月至2019年12月首都医科大学附属房山区良乡医院收治的患者109例为研究对象,均留取外周静脉血,测定血清抗-HCV抗体、HCV RNA,丙氨酸氨基转移酶(ALT)水平.结果 在收集的109例抗-HCV抗体阳性患者样本中,以HCV RNA为金标准作为丙型肝炎的诊断,HCV RNA阳性患者占20.2％(22/109),HCV RNA阴性患者占79.8％(87/109),两组样本经独立样本配对t检验,P值为0.006,差异有统计学意义(P<0.05),以HCV RNA检测为金标准,用SPSS 23.0软件对抗-HCV的S/CO值及ALT进行ROC曲线处理,HCV抗体S/CO值对丙型肝炎的ROC曲线下面积(AUC)为0.851,ALT对丙型肝炎的ROC曲线下面积(AUC)为0.831.结论 HCV RNA阳性患者ALT平均值明显高于HCV RNA阴性患者的ALT平均值.抗-HCV的S/CO值及ALT对于丙型肝炎的筛查都有一定的价值,且抗-HCV抗体优于ALT的筛查.临床上,患者筛查丙型肝炎抗体阳性时,可同时进行ALT的检测,可为丙型肝炎早期诊断提供依据,以免延误治疗时机.     

Necessity for Reassessment of Patients with Serogroup 2 Hepatitis C Virus (HCV) and Undetectable Serum HCV RNA

We encountered a patient positive for anti-hepatitis C virus (HCV) whose serum HCV RNA was undetectable with the Roche AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM) version 1 but showed a high viral load with the Abbott RealTime HCV assay (ART). Discrepancies in the detectability of serum HCV RNA were investigated among 891 consecutive patients who were positive for anti-HCV. Specific nucleotide variations causing the undetectability of HCV RNA were determined and confirmed by synthesizing RNA coding those variations. Serum samples with the discrepancies were also reassessed by CAP/CTM version 2. Among the 891 anti-HCV-positive patients, 4 patients had serum HCV RNA levels that were undetectable by CAP/CTM version 1 despite having levels of >5 log IU/ml that were detected by ART. All four patients had HCV genotype 2a and high titers of anti-HCV. Sequencing of the HCV 5′ noncoding regions revealed 2 common variations, A at nucleotide (nt) 145 and T at nt 151. Synthesized RNAs of the HCV 5′ noncoding region with standard (NCR145G151C) and variant nucleotides at nt 145 and nt 151 were quantified with CAP/CTM. RNAs of NCR145G151C and NCR145G151T were quantifiable with CAP/CTM version 1, while those of NCR145A151T and NCR145A151C went undetected. The substitution from G to A at nt 145 specifically conferred this undetectability, while this undetectability was reverted in synthesized HCV RNA with correction of this variation. Reassessment of these samples by CAP/CTM version 2 resulted in similar levels of HCV RNA being detected by ART. We conclude that HCV patients with undetectable HCV RNA by CAP/CTM version 1 should be reassessed for viral quantification.     

Impact of Isolated Hepatitis C Virus (HCV) Core-Specific Antibody Detection and Viral RNA Amplification among HCV-Seronegative Dialysis Patients at Risk for Infection

Amplification of hepatitis C virus (HCV) RNA from blood detected occult HCV infections in 30.9% of 210 HCV-seronegative dialysis patients with abnormal liver enzyme levels that had evaded standard HCV testing practices. Isolated HCV core-specific antibody detection identified three additional anti-HCV screening-negative patients lacking HCV RNA amplification in blood who were considered potentially infectious. Together, these findings may affect management of the dialysis setting.     

Restricted Isotypic Antibody Reactivity to Hepatitis C Virus Synthetic Peptides in Immunocompromised Patients

An enzyme immunoassay based on three synthetic peptides from the core, NS4, and NS5 regions of hepatitis C virus allowed the detection of antibodies in 100% of immunocompetent infected patients and in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). Immune impairment seemed to restrict the spectrum of antibody isotypes reacting to the core peptide.     

Abbott RealTime Hepatitis C Virus (HCV) and Roche Cobas AmpliPrep/Cobas TaqMan HCV Assays for Prediction of Sustained Virological Response to Pegylated Interferon and Ribavirin in Chronic Hepatitis C Patients

Two commercial real-time PCR assays are currently available for sensitive hepatitis C virus (HCV) RNA quantification: the Abbott RealTime HCV assay (ART) and Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM). We assessed whether the two real-time PCR assays were more effective than Roche Cobas Amplicor HCV Monitor test, v.2.0 (CAM) for prediction of the sustained virological response (SVR) to pegylated interferon (PEG-IFN) plus ribavirin (RBV) in chronic hepatitis C. Sixty patients chronically infected with HCV genotype 1b (37 males and 23 females, 53 ± 12 years of age) were treated with PEG-IFNα2b plus RBV for 48 weeks. Stored specimens at nine time points for each patient (at baseline, on treatment, and 24 weeks after treatment) were tested by the two real-time PCR assays and CAM. Twenty-six (43.3%) patients reached SVR. The positive predictive values (PPVs) for SVR of undetectable HCV RNA at week 12 by CAM, ART, and CAP/CTM were 74.3%, 88.0%, and 95.2%, respectively. An undetectable HCV RNA level by CAM, ART, and CAP/CTM correctly predicted SVR at week 4 in 100%, 100%, and 100% of patients, at weeks 5 to 8 in 91.7%, 100%, and 100% of patients, at weeks 9 to 12 in 55.6%, 75%, and 87.5% of patients, and at weeks 13 to 24 in 0%, 26.7%, and 40% of patients, respectively. Of 16 patients who relapsed after treatment, HCV RNA was detectable in 2 patients at the end of treatment by CAP/CTM but undetectable by ART and CAM. HCV RNA tests using ART and CAP/CTM are considered to be more effective at predicting SVR than CAM, and the PPV for SVR was slightly higher in CAP/CTM than in ART.     

一起甲型肝炎暴发疫情调查与控制分析

目的分析一起甲肝的暴发原因及控制措施。方法经过规范的流行病学调查,在SPSS13．0软件上对所得数据进行统计分析。结果有21名甲肝患者,其中19例有饮用生水史,2例未有饮用生水史（P〈0．001）。结论饮用水污染是本次甲肝暴发的原因,应该加强生活饮用水的安全检测,防止污染。     

An OPTIMIZE Study Retrospective Analysis for Management of Telaprevir-Treated Hepatitis C Virus (HCV)-Infected Patients by Use of the Abbott RealTime HCV RNA Assay

Protease inhibitor (PI)-based response-guided triple therapies for hepatitis C virus (HCV) infection are still widely used. Noncirrhotic treatment-naive and prior relapser patients receiving telaprevir-based treatment are eligible for shorter, 24-week total therapy if HCV RNA is undetectable at both weeks 4 and 12. In this study, the concordance in HCV RNA assessments between the Roche High Pure System/Cobas TaqMan and Abbott RealTime HCV RNA assays and the impacts of different HCV RNA cutoffs on treatment outcome were evaluated. A total of 2,629 samples from 663 HCV genotype 1 patients receiving telaprevir/pegylated interferon/ribavirin in OPTIMIZE were analyzed using the High Pure System and reanalyzed using Abbott RealTime (limits of detection, 15.1 IU/ml versus 8.3 IU/ml; limits of quantification, 25 IU/ml versus 12 IU/ml, respectively). Overall, good concordance was observed between the assays. Using undetectable HCV RNA at week 4, 34% of the patients would be eligible for shorter treatment duration with Abbott RealTime versus 72% with the High Pure System. However, using <12 IU/ml for Abbott RealTime, a similar proportion (74%) would be eligible. Of the patients receiving 24-week total therapy, 87% achieved a sustained virologic response with undetectable HCV RNA by the High Pure System or <12 IU/ml by Abbott RealTime; however, 92% of the patients with undetectable HCV RNA by Abbott RealTime achieved a sustained virologic response. Using undetectable HCV RNA as the cutoff, the more sensitive Abbott RealTime assay would identify fewer patients eligible for shorter treatment than the High Pure System. Our data confirm the <12-IU/ml cutoff, as previously established in other studies of the Abbott RealTime assay, to determine eligibility for shortened PI-based HCV treatment. (The study was registered with ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01241760","term_id":"NCT01241760"}}NCT01241760.)     

Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.     

一种简便快速筛选双位点单克隆抗体方法的建立

本文建立了一种无需对单克隆抗体(McAb)进行纯化和酶标记,利用市售酶标记抗体既可筛选双位点McAb的简易方法。其基本原理是,首先将待筛选McAb与酶标抗体制成McAb-酶标抗体复合物,再以此复合物作示踪剂进行夹心ELISA。若夹心孔OD492值高于第一、二阴性对照孔之和者, 提示所筛选McAb可能为双位点抗体。文中还对应用该方法时的注意事项进行了讨论。     

Definition of False-Positive Reactions in Screening for Hepatitis C Virus Antibodies

The rate of false-positive hepatitis C virus enzyme immunoassay results was determined to be at least 10% among 1,814 reactive serum samples based on (i) negative results in an independent confirmation assay, (ii) negative PCR results, and (iii) no patients developing clinical or biochemical signs of hepatitis during a 1-year follow-up.     

Multicenter Evaluation of the COBAS AMPLICOR HCV Assay, an Integrated PCR System for Rapid Detection of Hepatitis C Virus RNA in the Diagnostic Laboratory

The benefits shown by the recent introduction of PCR for the in vitro diagnosis of hepatitis C virus (HCV) infection has prompted the development of standardized, ready-to-use assays that can be implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV (COBAS), the first instrument system that allows the automation of HCV RNA amplification and detection, to determine its performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the results were compared with the results for biochemical and serological markers of HCV. In this study the two PCR systems showed the same accuracy, with a concordance rate of 99.8%. As expected, the correlation between serology and PCR was not absolute because the presence of anti-HCV antibodies may be associated with a latent or past infection. On the other hand, if the presence of confirmed anti-HCV antibodies and elevated alanine aminotransferase levels are taken as the “gold standard,” indicating an active, ongoing infection, the COBAS and AMPLICOR tests show high and comparable sensitivities (100%) and specificities (98%), with positive and negative predictive values of 100 and 97%, respectively. During the study no false-positive reactions were detected. The use of an internal control allowed the identification of inhibitory substances that prevented amplification for 0.3 and 0.4% of samples tested by the COBAS and AMPLICOR tests, respectively. Compared to the manual system, the COBAS system allowed a significant reduction of hands-on time and could improve the overall laboratory work flow. In conclusion, these results support the use of the COBAS and AMPLICOR tests for the molecular diagnosis of active HCV infections.