HCV core antigen plays an important role in the fight against HCV as an alternative to HCV‐RNA detection

ObjectiveTo discuss the clinical significance of HCV‐cAg testing in the diagnosis, activity determination, and monitoring of therapeutic effectiveness of HCV infection and its advantages compared with HCV‐RNA and anti‐HCV antibodies detection.MethodsBy summarizing the published literature, the advantages and significance of HCV core antigen detection were sought.ResultsThe expression of HCV‐cAg is highly consistent with that of HCV‐RNA, but compared with HCV‐RNA, detection of HCV‐cAg is easy to operate, time saving, and low cost. HCV‐cAg can be detected within 12~15 days after infection, and the window period can be shortened by5~7 weeks. HCV‐cAg is a serological indicator of virus replication, which can distinguish previous infection of HCV or current infection. HCV‐cAg detection is more suitable for immunocompromised, hemodialysis, organ transplant patients. HCV‐cAg also can be used to monitor antiviral efficacy and predict sustained virological response (SVR).ConclusionHCV core antigen has similar clinical sensitivity to NAT and can be used as a substitute for HCV‐RNA in the diagnosis of virus infection. Combined detection of HCV‐cAg and antibody serology can help doctors detect HCV infection earlier, accurately diagnose different stages of HCV infection, and evaluate the therapeutic effect of antiviral drugs, which are beneficial in the prevention and treatment of hepatitis C.     

Evaluation of saliva specimens as an alternative sampling method to detect hepatitis B surface antigen

In this study, a modified enzyme immunoassay (EIA) was evaluated for the Hepatitis B surface antigen (HBsAg) among whole saliva and oral fluid samples. Specimens were collected from 115 individuals who gave serum and oral fluid using Salivette (Sarstedt, Nümbrecht, Germany) and whole saliva. Saliva specimens were tested following a modified ELISA, and the results were compared with paired serum specimens that were tested according to the supplier's instructions. Transport buffer for the oral fluids, sample volume for assay, incubation period of sample with conjugate as well as cut-off values were evaluated to optimize the assay. The highest sensitivity and specificity were obtained by increasing the incubation of sample and conjugate to 16 hr and using the area under the receiver operating characteristic curve to calculate cut-off values. HBsAg was detected in 40 oral fluids and 44 whole saliva samples out of 47 paired positive serum specimens and not detected in 64 oral fluids and 63 whole saliva samples out of 68 matched negative sera samples by the ELISA assay. There was excellent agreement between the results for the serum and saliva specimens kappa value (κ): 0.80 for oral fluid and κ: 0.87 for whole saliva and there was excellent reproducibility. Using an optimized protocol, the sensitivities of whole saliva and oral fluid were 93.6 and 85.1%, respectively, whereas specificities of whole saliva and oral fluid were 92.6 and 94.1%, respectively. Our data showed a significant promise for the use of whole saliva and oral fluid together with the modified commercial EIA for Hepatitis B virus infection surveillance.     

A new HCV core antigen assay based on disassociation of immune complexes: an alternative to molecular biology in the diagnosis of early HCV infection

BACKGROUND: An EIA based on immune complex disassociation of nucleocapsid proteins of HCV has been developed to detect and quantify HCV core antigen. STUDY DESIGN AND METHODS: To evaluate whether this new assay (trak-C, Ortho Clinical Diagnostics) could be an alternative to NAT during the window period, its sensitivity in this context was assessed, and its performance was compared with that of a first-generation HCV core antigen assay dedicated to the blood screening (HCV core antigen ELISA). Studied populations included nine HCV RNA-positive, HCV antibody-negative blood donors and 23 hemodialysis patients who underwent an HCV seroconversion. From these individuals, 81 samples (23 HCV RNA-negative and 58 HCV RNA-positive) sequentially collected during the phase before seroconversion were tested. RESULTS: The nine blood donor samples were positive for the presence of HCV core antigen by the trak-C, and 6 of 8 tested were positive for the presence of HCV core antigen by blood screening ELISA. In the hemodialysis cohort, the 23 HCV RNA-negative samples were negative with the two HCV core antigen assays. Among the 58 HCV RNA-positive samples, 46 of 57 (80.7%) tested were positive for the presence of HCV core antigen with the blood screening assay, and 57 of 58 (98.2%) were positive for the presence of HCV core antigen with the trak-C. The mean delays in detecting HCV infection between trak-C and the appearance of HCV antibodies, between HCV RNA testing and trak-C, and between trak-C and HCV core antigen ELISA were 58.2, 0.24, and 3.33 days, respectively. CONCLUSION: Trak-C was more sensitive than the blood screening assay and had similar performance to HCV RNA assay in the window period. Trak-C could constitute an alternative to NAT for the diagnosis of HCV infection during the window period, especially when molecular biology procedures cannot be implemented.     

Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT

BACKGROUND: Preservation of the integrity of viral nucleic acids in blood specimens during shipping and handling is crucial for NAT and viral load monitoring. An economical and convenient method is described for nucleic acid stabilization by using an RNA stabilizing solution (RNAlater, Ambion) in plasma that is designed for the shipment of samples to tropical countries. STUDY DESIGN AND METHODS: HCV, HIV, and HBV FFP were compared with RNAlater-treated plasma and dried plasma spots (DPSs) after incubation at 37 degrees C, which was chosen as an upper limit of ambient shipping temperature, for up to 28 days. HCV-infected chimpanzee plasma was shipped at either room temperature after RNAlater treatment or as frozen plasma in liquid nitrogen from Liberia to New York City. They were then compared for HCV RNA levels. The nucleic acid stabilities were determined by quantitative PCR by using a molecular beacon assay on a sequence detection system (ABI 7700, PE-Biosystems) and by visualizing the PCR components on an acrylamide gel. RESULTS: Quantitative PCR data showed that a 60:40 or greater ratio of RNAlater:plasma volume successfully stabilized HCV RNA and HIV RNA in plasma for up to 28 days at 37 degrees C. HBV DNA in plasma was stable for up to 14 days at 37 degrees C without any stabilizing solution. DPSs on filter paper stabilized viral nucleic acids, but the recoveries were 3 to 10 times less than those with frozen plasma. The integrity of the 5' UTR region of HCV RNA in RNA later-treated chimpanzee plasma was intact when its PCR component was viewed on an acrylamide gel. CONCLUSION: The DPS method stabilized nucleic acids, at least with the extraction method used, was less sensitive than use of RNAlater, and required tedious manual handling. RNAlater provides a convenient way of stabilizing viral nucleic acid in plasma at ambient temperature during sample transportation.     

Sensitivity of HCV core antigen and HCV RNA detection in the early infection phase

BACKGROUND: Various countries have introduced HCV NAT to exclude infectious donations collected during the preseroconversion window phase (PWP). For the same purpose, an ELISA has also been developed to detect HCV core antigen (cAg). STUDY DESIGN AND METHODS: Using sequential samples from regular plasma donors with very recent HCV infections, a total of 494 samples from 52 anti-HCV-negative donors were collected. These panels were used for direct comparison of the performance of PCR and ELISA in detecting viral markers (RNA and cAg) during the PWP of HCV infection. The panels were genotyped, and each sample was analyzed by qualitative and quantitative HCV PCR and by cAg ELISA. The HCV RNA doubling time was calculated from quantitation of viral RNA in consecutive samples during the earliest outbreak of viremia. RESULTS: Concurrent detection of HCV RNA and cAg in 218 and nondetection in 185 samples yielded 81.6-percent concordance in the results of 494 samples. Unidirectional discrepancy of results (i.e., PCR positive and cAg negative) was seen in 91 of 494 (18.4%) samples, which was consistent with 65 specimens with RNA concentrations ranging between 300 and 100,000 IU per mL and 26 specimens with less than 300 IU per mL (limit of quantitative PCR). Individual genotyped panels had different kinetics and courses of viremia. The mean doubling time in the early PWP at the onset of viremia was derived to be 10.8 (range, 5.8-21.0) hours. CONCLUSION: A majority of HCV RNA-positive samples were also cAg-positive during the PWP. The current cAg detection corresponds to 100,000 IU per mL of HCV RNA. Since low-titer samples would be identified only by single-donation NAT, which is often affordable only in developed countries, the cAg ELISA could offer a practical alternative for some countries. The doubling time for HCV RNA at the onset of viremia corresponds to a calculated mean delay of cAg detection during the virus burst phase of 2 or 5 days, when compared with minipool (5000 IU/mL) or single-donation NAT (50 IU/mL), respectively.     

Efficacy of HCV core antigen detection during the preseroconversion period

BACKGROUND: The purpose of this study was to compare the performances of HCV core antigen (HCV Ag) testing with HCV RNA detection during the preseroconversion period. STUDY DESIGN AND METHODS: Six HCV antibody (HCV Ab)-negative and HCV RNA-positive blood samples from 6 donors and 135 serial samples from 28 patients who had undergone hemodialysis, collected a mean of 90 days before the detection of HCV Ab, were tested by ELISA for the detection of HCV Ag and by PCR to quantify HCV RNA. RESULTS: Five of the six donors were positive for HCV Ag. The donor with a negative HCV Ag test had the lowest viral load. In the hemodialysis patients, the 43 first specimens of the series were HCV RNA negative. Of the 92 specimens that were HCV RNA positive, 81 (88%) were positive for HCV Ag. Among the 74 samples with more than 10(5) RNA copies, 71 (96%) were HCV Ag positive. Average time from first viremic bleed to first HCV Ag-positive bleed was estimated at 2.0 days and that to first HCV Ab-positive bleed at 50.8 days. CONCLUSION: HCV Ag testing permits the detection of an HCV infection about 1.5 months earlier than the HCV Ab screening tests and an average of only 2 days later than quantitative HCV RNA detection in individual specimens.     

Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay

BACKGROUND: Currently, the detection of HCV infection in blood donors relies on the ability of immunoassays to detect circulating HCV antibodies. However, a significant delay exists between the time of infection and the development of antibodies. This delay (window period) can last up to 70 days. The introduction of NAT for the detection of HCV RNA has reduced this window period dramatically. However, NAT is labor intensive, prone to contamination, and expensive as compared with standard serologic tests. STUDY DESIGN AND METHODS: An automated, microparticle-based chemiluminescent assay for the detection of HCV core antigen in human serum and plasma was developed. The specificity and sensitivity of this prototype assay were evaluated by testing a population of normal blood donors and commercially available seroconversion panels. RESULTS: The HCV core antigen assay exhibited a 99.9-percent specificity by detecting a single repeatably reactive sample out of 1004 normal donors tested. Assay sensitivity was determined by comparing the HCV core antigen detection rate with the antibody seroconversion profile and the rate of HCV RNA detection. Among 15 seroconversion panels examined, core antigen was detected in 69 of 70 antibody-negative and/or RNA-positive samples for a sensitivity relative to NAT of 98.6 percent. CONCLUSION: These data indicate that the automated, microparticle-based chemiluminescent HCV core antigen assay can reduce the window period for detection of potentially infected blood donors by 32.7 days, and it represents a viable alternative to HCV RNA testing.     

探讨丙肝核心抗原检测在母婴传播中的应用价值

目的：探讨丙肝核心抗原检测在母婴传播中的应用价值。方法选取选取2014年1月～2015年1月在我院就诊的HCV‐Ab（ELISA）阳性产妇100例作为研究对象,产妇于生产前采集静脉血3～5 mL ,新生儿采集脐血2～3 mL 进行检测。首先使用HCV‐RNA 对100例HCV‐Ab（IGg）阳性产妇血清标本进行检测。使用HCV‐cAg、HCV‐Ab、HCVRNA三种方法对新生儿脐血标本进行检测,观察三种方法的阳性率。结果 HCVcAg阳性率与 HCV‐Ab（IgG）的阳性率比较差异具有统计学意义,P＜0．05。HCVcAg阳性率与HCV‐RNA阳性率比较,差异没有统计学意,P＞0．05。结论 HCVcAg检测在HCV母婴传播初期诊断中,可作为临床诊治的依据,值得基层医院推广。     

HCVRNA定量及HCV核心抗原和丙氨酸氨基转移酶结果分析

目的分析丙型肝炎病毒(HCV)RNA定量检测结果与HCV核心抗原及丙氨酸氨基转移酶(ALT)检测结果的相关性。方法收集涪陵监狱卫生所94例住院和门诊患者血清,采用逆转录聚合酶链反应荧光探针法定量检测标本中的HCV RNA,同时采用酶联免疫吸附试验检测HCV核心抗原,采用全自动生化分析仪检测ALT水平。结果 94例样本中HCV RNA阳性率为56.4%(53/94),HCV核心抗原阳性率为53.2%(50/94),经统计学分析,两种方法的阳性率差异无统计学意义(P>0.05),符合率为84.9%(45/53);ALT异常率为62.3%(33/53),随着HCV RNA含量的升高而增加。结论 HCV RNA定量检测结合HCV核心抗原及ALT检测,可帮助临床了解HCV在体内的复制水平及肝脏的炎性反应状态,以指导临床用药及疗效观察。     

High frequency of HCV infection in individuals with isolated antibody to hepatitis B core antigen

Although isolated antibody to hepatitis B core antigen (anti-HBc) is frequently nonspecific or may be the only serological marker of past self-limiting hepatitis B, where antibodies against the surface antigen have disappeared, isolated anti-HBc seropositivity is frequently associated with chronic hepatitis B in HIV- and HCV-infected individuals. Of 5,520 samples that tested positive for anti-HBc (IMx and AxSYM CORE, Abbott, Delkenheim, Germany) at the Institute of Virology, University Clinic Frankfurt during the time interval from January 1994 to February 1996, 643 (11.6%) were isolated anti-HBc-reactive in the IMx and AxSYM CORE assays (inhibition values >90%). There was a statistically significant association between isolated anti-HBc seropositivity and HCV and HIV/HCV coinfection (p < 0.05). A total of 190 samples were available for further testing. Six (3.2%) of 190 isolated anti-HBc-positive samples were considered false-positive since they were only positive in the AxSYM or IMx CORE assay and a linear decrease of the measured signal could not be observed in dilution series. Of 184 serum samples tested with nested PCR using primers of the S genome region, only 6 (3.3%) were HBV DNA-positive. Anti-HBc-IgM antibody could be detected in 3 (1.6 %) of the tested samples using the IMx CORE-M. With the more sensitive VIDAS HBc IgM specific IgM antibody was detected in 15 (8.5%) of 177 samples at concentrations ranging from 10 to >200 Paul Ehrlich Institute U/ml. HIV or HCV coinfection was present in 28.1% and 37.5% of isolated anti-HBc-positive individuals, respectively. We conclude from our observations that only a limited proportion of anti-HBc-isolated individuals are potentially infectious, however anti-HBc-IgM which is detectable in any form of liver disease associated with HBV infection was present in more than 8% of the individuals. Of isolated anti-HBc-positive sera 37% were positive for anti-HCV, suggesting that anti-HCV antibody testing should be performed in isolated anti-HBc-positive individuals.     

丙型肝炎病毒载量与核心抗原及丙氨酸转氨酶检测的比较研究

目的研究实时荧光定量PCR定量检测丙型肝炎病毒(HCV)RNA载量与HCV核心抗原和丙氨酸氨基转移酶(ALT)检测的相关性及符合性。方法逆转录-PCR荧光探针法定量检测94例怀疑HCV感染患者的血清HCV RNA,同时用酶联免疫吸附试验(ELISA)检测HCV核心抗原,全自动生化分析仪检测ALT水平。结果 94份样本中HCV RNA阳性率为56.4%(53/94),核心抗原阳性率为53.2%(50/94),经统计学分析,两种方法的阳性率差异无统计学意义(P>0.05),符合率为84.9%(45/53);ALT异常率为62.3%(33/53),并随HCV RNA载量的升高而增加。结论 HCV RNA定量检测及HCV核心抗原检测结合ALT结果分析有助于临床了解HCV在体内的复制水平和肝脏的炎性反应状态,指导临床用药及观察疗效。     

HCV核心抗原动态监测抗HCV疗效的临床研究(英文)

Objective To evaluate the performance of Hepatitis C virus (HCV) core antigen and HCV RNA PCR in the determining of the efficacy of HCV antiviral therapy in patients infected with HCV.Methods HCV core antigen and HCV RNA were measured in sera of 35 chronic HCV infected Chinese patients.Concentrations of HCV core antigen and HCV RNA were analyzed at 5 time points before,during and at the end of antiviral therapy.Results This study showed that the HCV core antigen and HCV RNA concentrations in 35 HCV patients were significantly correlated.Decrease of HCV core antigen and HCV RNA concentrations at the 4th,12th,24th and 48th week were observed during the antiviral therapy.However,HCV core antigen levels at week 12 and 24 of therapy were significantly lower than those at week 4 (P<0.05).In contrast,no further decrease was observed in HCV RNA concentrations at weeks 12 and 24 (P>0.05).HCV core antigen testing may be advantageous in some cases,in particular,the low levels of HCV core antigen at week 4 may be predictive of satisfactory outcome of treatment.Conclusions HCV core antigen represents a stable and sensitive marker of viral replication and could be used to monitor the clinical efficacy of HCV antiviral therapy.     

血清转换样本丙型肝炎病毒核酸、核心抗原及抗体检测对比研究

目的探讨丙型肝炎病毒(HCV)核心抗原酶联免疫诊断试剂检测早期HCV感染“窗口期”样本的可行性。方法用新研制的丙型肝炎病毒核心抗原(HCV-cAg)酶联免疫诊断试剂检测11份系列血浆转换样本的抗-HCV、HCV-cAg、HCV RNA,并与病毒核酸及抗体检测方法做对比研究。结果HCV-cAg较抗-HCV检出提前14~51d,平均32.7d;HCV RNA较抗-HCV检出提前19~51d,平均36.0d。结论HCV-cAg酶联免疫诊断试剂可用于HCV早期感染“窗口期”样本的检测,降低输血风险度。