Diagnostic significance of antibody detection to various hepatitis C virus antigens in patients with acute and chronic HCV-infection

AIM: To examine diagnostic value of antibodies to various HCV antigens in patients with acute and chronic HCV-infection. MATERIAL AND METHODS: Enzyme immunoassay has tested blood sera from 136 patients with icteric acute hepatitis C (AHC) and 45 patients with chronic HCV infection for IgG antibodies to antigens of proteins core, NS4, NS5, HCV. Synthetic peptides core-16, NS4-20, NS5-23 were used as antigens. RESULTS: Patients with icteric AHC had IgG antibodies to antigens of both structural protein core and non-structural proteins NS4, NS5 of HCV as early as the first 10 days of jaundice. Occurrence of anti-core and anti-NS4 increases with the disease duration. Incidence of anti-NS4 correlated with duration of previous intravenous drug addiction. In patients with AHC early in the icteric period anti-core, anti-NS4, anti-NS5 were present less frequently than in patients with chronic HCV infection having elevated levels of AlAT. Significant differences were found neither with the group with normal AlAt nor in the spectrum of the detected antibodies between patients with acute and chronic HCV infection. CONCLUSION: Despite different frequency of anti-core, anti-NS4, anti-NS5 detection in patients with icteric AHC and patients with chronic HCV-infection and high AlAT, their high incidence rate in this or that group and absence of differences by the spectrum of the studied antibodies do not allow the fact of their detection to be a diagnostic marker differentiating acute HCV-infection with chronic one.     

Detection of hepatitis C virus RNA in patients with chronic hepatitis C virus infections during and after therapy with alpha interferon.

In 24 patients with hepatitic C virus (HCV) infection who participated in a randomized trial with alpha 2B interferon, HCV RNA analysis by the polymerase chain reaction with two separate primer sets was performed at weeks 0, 4, and 24 and during a follow-up period of 6 to 9 months. Prior to therapy all patients were HCV RNA positive. During therapy, HCV RNA decreased to an undetectable level (< 1 chimpanzee infectious dose per ml) in nine patients at week 4. After week 4, no additional cases of HCV RNA disappearance (< 1 chimpanzee infectious dose per ml) were observed. During follow-up, HCV RNA could not be detected in four of the six patients with a sustained alanine aminotransferase response. These results suggest the probable predictive value of HCV RNA levels for detecting the failure of therapy in an early stage of HCV infection.     

福建省慢性丙型肝炎患者丙型肝炎病毒基因分型的分析

目的观察福建省慢性丙型肝炎患者基因型分布特点以及与患者性别、年龄、丙型肝炎病毒（HCV） RNA 之间的关系。方法应用反转录聚合酶链反应（RT-PCR）和 SANGER 测序法对155例慢性丙型肝炎患者的HCV 进行基因分型。结果在155例标本中,HCV 基因1型占55．48％,基因2型占18．71％,基因3型13．51％,基因6型12．26％,未见基因4、5型;男女慢性 HCV 感染均以1型为主要基因型,在其他基因型中,男性3型、6型多于2型,女性则以2型为主,差异有统计学意义（P ＜0．05）;基因2型平均年龄为（49岁）,3型平均年龄为（35岁）,差异有统计学意义（P＜0．05）。结论福建地区 HCV 基因型以1b 型为主,其次是2a 型,未见基因4、5型;基因2型的感染者平均年龄较基因3型大。     

HBV RNA检测在慢性乙型肝炎中的临床价值

目的对检测乙型肝炎病毒(HBV)RNA的方法进行评估,比较不同乙型肝炎血清标志物模式患者血清HBV RNA水平及其与HBV DNA之间的相关性。方法对HBV RNA检测方法的精度密、灵敏度及线性范围进行评估。收集81例乙型肝炎表面抗原(HBsAg)阳性慢性乙型肝炎患者的血清,同时检测HBV RNA、HBV DNA及乙型肝炎血清学标志物。按乙型肝炎e抗原(HBeAg)是否阳性、不同乙型肝炎血清标志物模式及是否接受抗病毒药物治疗分别分组。结果 HBV RNA高值、低值样本的批内精密度分别为0.56%、2.30%,批间精密度分别为3.13%、6.03%,均符合临床要求;对接近厂商声明的检测下限的样本重复检测20次,检出率为100%,灵敏度符合要求;在HBV RNA 1.0×10²～1.0×10⁷拷贝/mL范围内成线性。HBeAg阳性组血清HBV RNA和HBV DNA载量均高于HBeAg阴性组(P<0.01、P<0.05)。在不同乙型肝炎血清标志物模式中,HBeAg阳性且乙型肝炎e抗体(HBeAb)阴性患者血清HBV RNA载量最高(P...     

The role of physician extenders in managing patients with chronic hepatitis C

The number of "physician extenders" (nurse practitioners and physician assistants) caring for patients with chronic hepatitis C is rising rapidly. Their growing role in the management of these patients promises greater efficiency in the delivery of care and more provider interaction with patients. This may yield benefits in terms of patient education and support, management of medication side effects, and patient adherence to treatment regimens. This article reviews the role of physician extenders in the management of patients with hepatitis C and outlines strategies for maximizing their contribution to the care of these patients.     

Immune parameters in patients with chronic hepatitis C and with various histologic changes

Levels of cytokines (IL-1, IL-2, IL-4, IL-6, TNF) were studied in 22 patients with chronic hepatitis C (CHC) under different value of histological activity index (HAI). Cytokines were measured in peripheral blood by enzyme immunoassay using kits produced by Proteinovy Kontur (St-Petersburg). Morphological examination of hepatic biopsies was made with estimation of HAI by R. Knodell and fibrosis degree by V. Desmet. CHC patients with a high HAI were found to have higher levels of gamma-globulines and gamma-GTP. No significant correlation was revealed between HAI and content of transaminases. CHC patients with definite inflammatory infiltration of the portal tracts, intralobular degeneration, hepatic necrosis had higher indices of proinflammatory mediators of immune response IL-1, IL-2, IL-6 and TNF-alpha. In severe hepatic fibrosis there were higher values of IL-1, IL-2, IL-6. IL-4 was higher in patients with weak inflammatory infiltration, intralobular degeneration, lobular necrosis, mild fibros. The latter was associated with high interferon-alpha. Thus, serum cytokines concentrations and morphological changes in the liver correlate.     

慢性HBV感染者肝组织HBV cccDNA的定量检测

目的建立一种肝活检组织中HBV共价闭合环状DNA（cccDNA）的定量检测方法。方法待检肝组织标本共21份，来源于江苏省人民医院肝脏手术患者，包括19份慢性HBV感染，其中HBeAg（＋）标本10份，HBeAg（-）标本9份，4份非HBV感染为阴性对照组，取HBVDNA阳性的患者外周血作为rcDNA组。检测方法的主要步骤为肝组织经蛋白酶K和细胞裂解缓冲液消化后，用液相抽提法提取核酸，将提取的核酸溶液分为2等份。1份用特异性降解单链DNA的核酸酶加以消化，纯化后使用跨缺口引物和SYBR GreenⅠ荧光染料进行实时荧光定量PCR分析；另1等份则用以定量检测肝细胞看家基因（β-Globin）作为样本细胞参数。检测结果的特异性主要通过PCR反应产物的序列分析及rcDNA组结果的对照进行证实，HBeAg（＋）组和HBeAg（-）组cccDNA水平的差异通过两样本t检验进行分析。结果PCR产物经琼脂糖凝胶电泳分析显示扩增产物的碱基数为350bp左右，DNA测序分析提示产物与目的片段的序列符合率为99％以上，且以rcDNA为对照的结果均为阴性，排除最有可能造成假阳性的rcDNA对结果的干扰。本方法对10mg HBeAg（＋）肝组织标本的cccDNA的检测阳性率为100％。血清HBeAg（＋）的肝组织样本的平均HBV cccDNA水平高于HBeAb（＋）的肝组织标本（P〈0．05）。结论通过上述三种途径证实了本文所建立方法的特异性。应用SYBR GreenⅠ荧光染料和β-Globin作为样本细胞参数所建立的实时荧光定量PCR方法检测肝细胞内HBV cccDNA，具有较高的特异性、敏感性，且成本较低的特点。     

Phylogenetic analysis of hepatitis GB virus type C/hepatitis G virus in Spanish patients with chronic hepatitis B or C virus infection.

The nucleotide sequence of hepatitis GB virus type C (HGBV-C)/hepatitis G virus (HGV) NS3/helicase and 5'-untranslated regions from 23 Spanish patients were analyzed to assign the HGV isolates one of the proposed HGBV-C/HGV genotypes. The analysis of the evolutionary distance frequency showed that the distances among all sequences in NS3/helicase region were distributed around a single peak of 0.20, suggesting that all included sequences belonged to the same HGBV-C/HGV genotype. By contrast, in the 5'-untranslated region, all the distances corresponding to our sequences and those of the HGBV-C/HGV types 2 and 3 were distributed around a major peak of 0.03. The remaining distances corresponding to the HGBV-C/HGV type 1 sequences were distributed around a minor peak of 0.11. The phylogenetic tree and pairwise comparison of evolutionary distances among the 5'-untranslated region of the infected patients and each HGBV-C/HGV genotype demonstrated that our HGBV-C/HGV isolates belonged to subtype 2a (17/23; 78%) and 2b (5/23; 22%). No relation was found between HGBV-C/HGV subtype and hepatitis B or C virus infection.     

甲状腺过氧化物酶抗体检测对慢性丙型肝炎患者的临床意义

目的 检测慢性丙型肝炎患者甲状腺过氧化物酶抗体的临床意义.方法 对健康对照组62例,慢性乙型肝炎患者组68例,慢性丙型肝炎患者组74例,分别检测甲状腺过氧化物酶抗体(TPOAB);对慢性丙型肝炎患者检测丙氨酸氨基转移酶(ALT)、抗核抗体(ANA)、抗线粒体抗体(AMA)及类风湿因子(RF).结果 慢性丙型肝炎患者...     

Removal of hepatitis C virus by G-1 beads in sera from patients with chronic hepatitis C

Recently, a new method of extracorporeal granulocyte depletion apheresis has been developed to treat inflammatory systemic diseases using an Adacolumn (Japan Immunoresearch Laboratories, Takasaki, Japan) that is filled with acetate cellulose beads (G-1 beads) to adsorb the granulocytes. We examined whether hepatitis C virus (HCV) is adsorbed after incubation of the Adacolumn with the sera from patients with HCV-RNA-positive chronic hepatitis C. PATIENTS AND METHODS: A total of 10 patients with chronic hepatitis C, whose levels of HCV RNA were greater than 800 kIU/ml were examined. The serum was incubated with 500 G-1 beads in a syringe at 37 degrees C for 1 h. After removal of the serum, the beads were washed with RNase-free water. The G-1 beads were removed from the syringe after centrifugation. RNA was extracted from 200 microl of the wash waste and from 10, 50, 100 and 200 beads, respectively, using TRIZol regent. Detection of HCV RNA was performed using the nested PCR method. RESULTS: HCV RNA was detected from as few as 10 G-1 beads. HCV RNA was not detected from waste fluid collected after the last wash from any of the patients. Further, HCV RNA was detected in the initial waste fluid after the 37 degrees C incubation with serum in all of the patients. Since HCV RNA was detected on the G-1 beads, but not from the last washing solution in the current examination, these results suggest that the G-1 beads adsorbed HCV RNA. CONCLUSIONS: Our in vitro study confirmed that G-1 beads adsorbed HCV; therefore, apheresis using a column filled with G-1 beads may reduce the HCV RNA load in the blood of patients with chronic hepatitis C.     

慢性丙型肝炎患者病毒基因分型及HCV RNA相关性研究

目的：探讨江苏地区慢性丙型肝炎患者病毒基因分型特点及其与患者年龄、性别和丙型肝炎病毒（HCV）RNA水平的关系。方法收集抗 HCV 阳性血清1032份,采用荧光定量 PCR（Q‐PCR）法检测患者血清HCV RNA水平,HCV基因分型芯片进行基因分型检测,应用SPSS19．0软件对数据进行统计分析。结果检测出HCV RNA阳性样本704份,其中 HCV 单基因型674份,1b、2a、3b、3a、1a 和6型分别占75．71％、7．95％、7．95％、1．99％、0．99％和1．14％;混合基因型30份,1b／2a、1b／3b、1b／3a和2a／3b型分别占2．27％、1．56％、0．28％和0．14％。不同基因型均以男性和青壮年居多,且病毒载量差异有统计学意义：1b和3a相近,都高于2a和1b／2a病毒载量。结论江苏地区 HCV基因型以1b型为主,其次是2a、3b型。慢性丙型肝炎患者以中青年男性居多,而且不同 HCV基因型间病毒载量有差异。     

Direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles

BackgroundGold nanoparticles (AuNPs) exhibit a unique phenomenon known as Surface Plasmon Resonance, which is responsible for their intense red color. This color changes to blue upon aggregation of AuNPs.ObjectiveThis work aims to develop a rapid, simple and cheap assay for direct detection of unamplified HCV RNA extracted from clinical samples using unmodified AuNPs.MethodsSerum samples were collected from healthy volunteers (n = 45) and chronic HCV patients (n = 30). Extracted RNA, hybridization buffer containing PBS, and a primer targeting the 5′UTR of HCV were mixed. The mixture was denatured, annealed, and then cooled to room temperature for 10 min followed by addition of AuNPs.ResultsSalt, primer, AuNPs concentrations and annealing temperature and time were all optimized. In HCV positive specimens, the color of the solution changed from red to blue within 1 min. The assay has a sensitivity of 92%, a specificity of 88.9%, and a detection limit of 50 copies/reaction.ConclusionsTo our knowledge, this is the first assay that allows the detection of unamplified HCV RNA in clinical specimens using unmodified AuNPs. The developed assay is highly sensitive, has a turnaround time of 30 min, and eliminates the need for thermal cycling and detection instruments.     

慢性丙型肝炎患者血清基因型与HCV RNA的相关性研究

目的 研究丙型肝炎患者HCV基因型的分布,探讨基因型在性别上的分布以及基因型与HCV RNA病毒载量的相关性.方法 收集2010年5-12月来自40家医院的206例丙型肝炎患者的血清标本,采用瑞士罗氏公司生产的定量PCR试剂(罗氏试剂)对进行HCV RNA检测,应用雅培公司生产的Abbott RealTime HCV GenotypeⅡ试剂(雅培试剂)对206例丙型肝炎患者的血清标本进行基因分型,分析基因型在性别上的分布以及HCV基因型与HCV RNA病毒载量的相关性.结果 206份HCV RNA阳性血清标本中HCV1型(未具体分1a和1b型)占3.4%(7/206)、1a型占1.0%(2/206)、1b型占59.7%(123/206)、2型占15.5%(32/206)、3型占13.1%(27/206)、6型占2.9%(6/206)、1/6混合型占2.4%(5/206)、2/4混合型占0.5%(1/206),未分型占1.5%(3/206).132例基因1型和65例非基因1型(2型、3型和6型)患者HCV基因型在性别上的分布差异无统计学意义(x2=0.000,P＞0.05).188例患者不同基因型之间血清HCV RNA病毒载量差异有统计学意义(F=3.371,P＜0.05).将197例HCV单基因型患者按地区分为东、南、西、北、中5组,基因型1型与非基因1型在地区分布上差异无统计学意义(x2=5.840,P＞0.05).结论 丙型肝炎病毒感染以1b型为主,其次为基因2型.基因型在性别上的分布没有差异.基因1b型HCV RNA病毒载量高于基因3型,基因2型HCV RNA病毒载量高于基因3型,基因6型HCV RNA病毒载量高于基因3型.     

Detection of replicative intermediates of hepatitis C viral RNA in liver and serum of patients with chronic hepatitis C.

The hepatitis C virus is a positive stranded hepatotropic RNA virus. We describe a method of detecting positive and negative strands of hepatitis C viral RNA using the polymerase chain reaction. We tested serum and liver tissue from nine patients with chronic hepatitis C. The positive RNA strand of HCV was detected in the sera and livers of all nine, the negative strand was detected in the livers of eight (89%), and in the sera of five (55%). Titers of both strands of HCV RNA were determined by serial endpoint dilutions. The amount of the negative strand in the serum and liver was usually 10-100 times less than the positive strand. Predigestion of serum with ribonucleases did not alter the detection of the negative strand. This suggests that the negative strand found in the serum may be protected from digestion by being associated with virions.     

慢性丙型肝炎第一高变区抗体与HCV RNA病毒滴度相关性的研究

[目的]探讨慢性丙型肝炎患者第一高变区抗体与HCVRNA滴度的关系。[方法]采用计算机软件辅助分析确定27条HVR1基因序列，并克隆表达了系列重组HVR1抗原。应用27条不同的HVR1抗原包被酶联板，采用间接ELISA法测定慢性丙型肝炎患者样品HVR1抗体，并利用定量RT—PCR对病人血样完成HCVRNA测定，根据测定HCVRNA病毒滴度分为6组，10^2、10^3、10^4、10^5、10^6、10^7，每组6人，分别与27条不同的HVR1抗原进行反应，测定抗HVR1抗体，分别计算每组每种HVR1抗原的OD平均值。[结果]10^2、10^3、10^4组对27条HVR1抗体反应OD平均值均低于10^5、10^6、10^7组，而10^5组对27条HVR1抗体反应OD平值均最高。随着HCV RNA滴度的升高，27条HVR1抗体反应OD平均值略有下降。[结论]慢性丙型肝炎患者多种HCV第一高变区抗体与HCVRNA病毒滴度有一定的关系。     

Danoprevir monotherapy decreases inflammatory markers in patients with chronic hepatitis C virus infection

Danoprevir is a potent and selective direct-acting antiviral agent that targets the protease activity of hepatitis C virus (HCV) NS3/4A. This agent results in a significant rapid decline in HCV RNA levels when it is used in monotherapy. The present study evaluated whether plasma concentrations of the inflammatory markers gamma interferon-inducible protein 10 (IP-10) and neopterin or the interferon-stimulated gene product 2'-5'-oligoadenylate synthetase (OAS-1) were correlated with the plasma HCV RNA concentration before or during 14-day danoprevir monotherapy. In contrast to pegylated interferon and ribavirin treatment, a higher baseline IP-10 concentration was positively correlated with a greater first-phase HCV RNA decline upon danoprevir administration. Changes in the IP-10 plasma concentration during danoprevir administration were also associated with categorical changes in HCV RNA concentration at days 7 and 14. The neopterin concentration appeared to be moderately decreased during danoprevir administration, although these changes were not statistically significant. However, changes in neopterin concentration showed a statistically significant correlation with changes in IP-10 concentration. Considerable variation in the OAS-1 concentration was observed before and during treatment, including in patients treated with placebo and/or patients with minimal virologic response. Overall, these results suggest that effective treatment with a direct-acting antiviral agent may reduce hepatic inflammation and that first-phase HCV RNA decline during treatment with an NS3/4A protease inhibitor is more robust in patients with high baseline IP-10 concentrations.