Quantitative assessment of hepatitis C virus RNA in peripheral blood mononuclear cells during therapy with interferon-α2a

A significant number of patients with hepatitis C (HCV) treated with interferon (IFN) will initially clear their serum of HCV RNA, but will then have recurrence of viraemia either during or after therapy. One proposed mechanism for relapse is that HCV may persist in peripheral blood mononuclear cells (PBMCs) and that the PBMCs serve as a 'viral reservoir' that is resistant to IFN. To address this hypothesis, we performed serial, quantitative polymerase chain reaction (PCR) of HCV RNA in serum and PBMCs from 26 consecutive patients treated with IFN-α2a. Of the 26 patients, 11 (42%) did not clear virus from their serum during therapy and were termed non-responders. Five patients (19%) had sustained clearance of virus from serum and were termed complete responders. The remaining 10 patients (39%) initially eliminated HCV RNA from their serum, but had relapse of viraemia. They were termed partial responders. In all 10 partial responders HCV RNA was undetectable in PBMCs at the same time that it was undetectable in serum. When virus recurred in serum, it was preceded by or occurred at the same time as the return of virus in PBMCs. The results of our study indicate that PBMCs did not serve as an IFN-resistant 'viral reservoir' during therapy. Partial responders who transiently cleared virus from serum also cleared virus from PBMCs and the presence or titre of HCV RNA in PBMCs at the initiation of therapy did not predict response to therapy.     

Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C

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实时荧光定量RT-PCR检测血清和单个核细胞中HCV RNA的意义

目的探讨实时荧光定量聚合酶链反应(FQ-RT-PCR)检测血清和外周血单个核细胞(PBMCs)中HCV RNA含量及其临床价值。方法采集可疑丙型病毒性肝炎病人血,用FQ-RT-PCR分别检测血清和PBMCs中HCV RNA含量。血清或PBMCS HCV RNA阳性者都视为HCV RNA阳性。结果检测可疑患者180例,HCV RNA阳性106例。其中血清HCV RNA阳性84例,占79.25%;PBMCs HCV RNA阳性63例,占59.43%。HCV RNA阳性比率血清高于PBMCs(χ2=9.78,P<0.01)。单独血清HCV RNA阳性43例,占40.57%;单独PBMCs HCV RNA阳性22例,占20.75%;血清和PBMCs中HCV RNA同时阳性41例,占38.68%。血清和PBMCs HCV RNA含量差异无显著性。结论同时检测血清和PBMCs HCV RNA可提高HCV感染诊断的阳性率,检测PBMCs HCV RNA对抗病毒治疗的疗效评价及治疗时间有重要意义。     

Intracellular levels of hepatitis B virus DNA and pregenomic RNA in peripheral blood mononuclear cells of chronically infected patients

Summary.  It remains uncertain whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA) can be detected in the serum or peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) infection. We examined HBV cccDNA and pgRNA in the serum and PBMC, and investigated the effect of lamivudine therapy on the viral loads in the PBMC of CHB patients. Paired serum and PBMC samples from 50 treatment-naïve CHB patients [25 hepatitis B e antigen (HBeAg) positive and 25 HBeAg negative] were quantified for total HBV DNA, cccDNA and pgRNA by real time polymerase chain reaction. HBV cccDNA and pgRNA were below the lower detection limit in all serum samples, and in 84% of PBMC. HBV DNA ( r  = 0.889, P  <   0.001) and pgRNA ( r  = 0.696, P  <   0.001) in PBMC correlated with the HBV DNA in serum. In the longitudinal study, 30 patients treated with lamivudine therapy for a median duration of 34 weeks (range 12–48 weeks) were examined. The median HBV DNA reduction in PBMC before and after treatment was 1.318 (range −0.471 to 3.846) log units, which was significantly lower than serum HBV DNA reduction [3.371 (range −0.883 to 9.454) log units, P  <   0.05]. HBV cccDNA and pgRNA were undetectable in the serum of CHB patients. HBV viral loads in PBMC correlated with serum HBV DNA. Lamivudine therapy had less effect on the HBV viral loads in PBMC compared with the serum viral loads.     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒检测及其意义

目的 探讨外周血单个核细胞（ＰＢＭＣｓ）在丙型肝炎病毒（ＨＣＶ）的感染中的作用。方法 对２２例慢性丙型肝炎患者２１例抗－ＨＣＶ（＋）血液管析患者及１２例健康献血员的ＰＢＭＣｓ分别进行ＨＣＶＲＮＡ,ＨＣＶ抗原检测及电镜观察。结果 （１）２２生丙型肝炎肝炎患者ＰＢＭＣｓ中有７７．３％（１７／２２）ＨＣＶＲＮＡ阳性,（２）感染ＨＣＶ的ＰＢＭＣｓ中电镜下发现复制的ＨＣＶ颗粒;（３）ＨＣＶ颗粒阳笥者的血清和     

慢性丙型肝炎患者外周血单个核细胞中HCV RNA和NS5抗原的检测

目的 研究丙型肝炎病毒（ＨＣＶ）感染外周血个核细胞（ＰＢＭＣ）的情况及其意义。方法 运用非核素原位杂交法（ＮＩＳＨ）和抗生蛋白链菌素－生物素法（ＳＡＢＣ）分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ ＲＮＡ和ＮＳ５抗原。结果 ２０例患者中有８例（４０％）,ＰＢＭＣ中ＨＣＶ ＲＮＡ呈阳性,其中６例（３０％）ＰＢＭＣ中ＮＳ５抗原同时呈阳性。ＨＣＶＲＮＡ主要分布于胞浆中,而ＮＳ５抗原还可以出现在胞膜     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒感染研究

目的 以分子生物学、免疫学有电镜等技术对慢性丙型肝炎患者ＰＢＭＣｓ进行综合检测,以证实其受ＨＣＶ感染,并试图在电镜下发现和证实感染细胞内ＨＣＶ颗粒。方法 对逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学等技术分别检测患者ＰＢＭＣｓ内的ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,以常规和免疫电镜技术观察研究感染细胞内ＨＣＶ的形态、定位和超微结构。结果 ＨＣＶ ＲＮＡ阳性检出率为７７．２％（１７／２２）,ＨＣＶＡｇ     

原位杂交法检测外周血单个核细胞中HCV RNA

目的比较慢性丙型肝炎患者用干扰素治疗前以及治疗后３个月ＰＢＭＣ中ＨＣＶＲＮＡ。方法应用地高辛素标记ＨＣＶＲＮＡ正链及负链探针，建立原位杂交方法检测外周血单个核细胞（ＰＭＢＣ）中的ＨＣＶＲＮＡ。结果治疗前１９例患者正链ＨＣＶＲＮＡ阳性，８例负链ＨＣＶＲＮＡ阳性，用正链探针杂交在较多的细胞中出现杂交信号，负链探针杂交仅在少数细胞中出现杂交信号，ＨＣＶＲＮＡ在ＰＢＭＣ胞浆中呈均质性分布。用干扰素治疗结束后３个月２０例患者中９例ＨＣＶＲＮＡ转阴性，近期治愈率４５％。结论原位杂交技术的敏感性及特异性较高，且重复性较好，是研究ＨＣＶＲＮＡ在组织中定位分布和病毒复制场所一种切实可行的方法     

慢性乙型肝炎患者外周血单个核细胞内HBV DNA存在的意义

目的探讨外周血单个核细胞（PBMC）内HBVDNA与HBV标志物的关系及在慢性肝病发展过程中的作用．方法应用RCR技术结合斑点杂交技术测定血清HBVDNA及PBMC内HBVDNA血清学标志物采用ELISA法结果在HBsAg＋，HBeAg十组48例中，血清与PBMC内HBVDNA阳性检出率分别为93．8％和83．3％；在HBsAg＋，抗-HBe 组41例中，其阳性检出率分别为48．8％和58．5％.两组间存在显著性差异（P＜0．01）在其他各组中血清及PBMC内HBVDNA检出率均较抵有6例呈单纯PBMC内HBVDNA阳性.在不同类型慢性乙肝患者中，CSH和CMoH组PBMC-HBVDNA检出率分别为78．6％和61．8％，与CMiH组（25．0％）、ASC组（14．3％）比较，存在非常显著性差异（P＜0．01），呈随病情加重而检出率增高的趋势结论PBMC清除HBVDNA较血液缓慢，在病毒持续感染及造成肝细胞损伤过程中可能起一定作用．     

Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

Interferon (IFN)-α is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-α subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-α2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-α subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-α mRNAs and in the production of IFN-α protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-α5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-α8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-α subtypes, IFN-α5 being the specific subtype induced in PBMC from patients with chronic HCV infection.     

庚型肝炎病毒感染的临床特点及对外周血单个核细胞感染的研究

目的 探讨庚型肝炎病毒（HGV）感染的临床特点及其在体内的亲嗜性和复制位点。方法 采用逆转录－套式聚合酶链反应（RT-nested PCR)对129例高危人群的血清及其中59例的外周血单个核细胞(PBMCs)进行HGV RNA正、负链检查、临床分析。结果 9例单纯HGV感染者中,急性肝炎3例,慢性肝炎3例,另3例为无症状携带者;HGV对PBMCs的感染率（35．7％）显著低于丙型肝炎病毒（HCV）感染（77．3％,P＜0．05）;全部标本中均未发现HGV RNA负链存在。结论 HGV感染可引起急、慢性肝炎,但临床症状轻;HGV可感染PBMCs,但较HCV感染能力弱;HGV不在PBMCs中复制。     

Ligase chain reaction (LCR®) assay for semi-quantitative detection of HBV DNA in mononuclear leukocytes of patients with chronic hepatitis B

Summary. A ligase chain reaction (LCR®)-based approach to detect hepatitis B virus (HBV) DNA in peripheral blood mononuclear cells (PBMC) is described. Using this new amplification technique, we determined semi-quantitatively the amount of a short HBV S-gene fragment in PBMC lysates of 25 patients with different forms of chronic hepatitis (group A ( n = 8), hepatitis B s antigen (HBsAg)+/hepatitis B e antigen (HBeAg)+; group B ( n = 9), HBsAg+/HBeAg-; group C ( n = 8), HBsAg-/HBeAg-). The LCR results were compared with the findings obtained with polymerase chain reaction (PCR) amplification of three distinct HBV gene regions (preS1/2, S and C) and related to the serological profiles of the patients. Depending on the primer pair used for PCR amplification, sensitivity of HBV LCR in PBMC was equivalent or slightly superior to PCR. The highest positivity rate for HBV DNA was observed in the HBeAg+ and HBV DNA seropositive group (8/8) and was lower in the other patient groups B (4/9) and C (1/8). Interestingly, HBV gene sequences could also be detected in the lymphocytes of an HBsAg negative patient and in two patients from group B who were both negative for serum viral particles by PCR. The rapid LCR® procedure represents a reliable alternative to PCR for the sensitive detection of HBV DNA in PBMC samples. In combination with the automated IMx™-system the new amplification technique may be routinely used for screening for HBV in whole blood samples and thus may help to better evaluate the risk of HBV reinfection in liver transplant recipients.     

Hepatitis C virus RNA and long-term response to recombinant interferon-α2b in patients with chronic hepatitis C

Summary. The effectiveness of recombinant interferon-α2b (rIFN-α2b) in eradicating hepatitis C virus (HCV) RNA from serum has not been completely assessed. We studied 39 patients with compensated chronic hepatitis C diagnosed by liver biopsy and positive HCV RNA measured by polymerase chain reaction (PCR). Group I consisted of 26 patients treated with 3 MU of rIFN-α2b for 6 months; group II, 13 control patients observed for six months; and group III, 12 out of 13 patients from group n who subsequently received 5 MU of rIFN-α2b for 6 months. In group I, 11 out of 23 (47.8%) patients who completed treatment had an immediate response and five (21.7%) had a sustained response to therapy six months after treatment. No response was observed in patients from group II. In group III, 7 out of 12 (58.3%) patients who completed treatment had an immediate response and none had a sustained response. Considering all patients who completed rIFN-α2b treatment, HCV RNA remained positive at the end of therapy in three of five sustained responders (60%), six of 13 patients who relapsed (46.1%), and in all non-responders (100%). HCV RNA was positive 6 months after therapy in four (80%), 13 (100%). and 17 (100%) patients respectively. All patients with a sustained response had normal aminotransferase levels 18 months after therapy. We conclude that in chronic hepatitis C rIFN-α2b causes a significant immediate response but this is not sustained, only 2.8% of treated patients had a sustained loss of HCV RNA. Normal aminotransferase persist in the long term, despite persistence of HCV RNA.     

乙肝患者肝组织及外周血单个核细胞HBV DNA检测

用PCR方法对148例乙肝患者的肝组织及外周血单个核细胞HBV DNA进行检测，同时对肝组织进行病理组织学诊断。结果显示：148例乙肝患者的肝组织与PBMC内HBV DNA的检出率有一致性倾向，PBMC内HBV DNA的检出率与肝组织的损害程度呈正相关。     

Renal hepatitis C in the absence of detectable serum or hepatic virus

We report an apparently unique case where hepatitis C virus (HCV) RNA was identified in renal tissue from a patient with membranoproliferative glomerulonephritis and treated chronic hepatitis C, despite the absence of detectable virus in the serum or liver (COBAS Amplicor qualitative assay, lower limit of detection 50 IU/ml). The implications of this finding are discussed, with particular reference to current concepts regarding ‘occult’ hepatitis C infection.     

Distribution of different hepatitis C virus genotypes in patients with hepatitis C virus infection

AIM:To investigate the presence of mixed infection and discrepancy between hepatitis C virus(HCV) genotypes in plasma,peripheral blood mononuclear cells(PBMCs),and liver biopsy specimens.METHODS:From September 2008 up to April 2009,133 patients with chronic hepatitis C referred to Firouzgar Hospital for initiation of an antiviral therapy were recruited in the study.Five milliliters of peripheral blood was collected from each patient and liver biopsy was performed in those who gave consent or had indications...     

Correlation of serum HCV RNA and alanine aminotransferase levels in chronic hepatitis C patients during treatment with ribavirin

to evaluate the effect of ribavirin on serum hepatitis C virus (HCV) RNA and alanine aminotransferase (ALT) levels, 22 patients with chronic HCV infection were treated with oral ribavirin 1200 mg daily in three divided doses for 4 weeks. At the end of 4 weeks treatment, the serum ALT decreased in all but one patient and became normal in three individuals. The mean pretreatment serum ALT was reduced significantly from 193 ± 45 i.u./L to 95 ± 16 i.u./L after 4 weeks therapy (P= 0.009). However, 8 weeks after cessation of treatment, the serum ALT rose to a mean value of 154 ± 21 i.u./L. The mean pretreatment serum HCV RNA was not significantly decreased at the end of 4 weeks treatment (7.0 × 10⁵vs 4.1 × 10⁵ copies/mL, P > 0.05). However, serum HCV RNA levels were decreased in 12 and increased in 10 patients at the end of 4 weeks therapy. Eight weeks after cessation of therapy, the serum HCV RNA of 22 patients rose to a mean value of 4.9 ± 10⁵ copies/mL. Six patients who continued to have elevated serum ALT and positive HCV RNA after the initial 4 weeks treatment received oral ribavirin at the same dosage for an additional 24 weeks. The serum ALT again decreased in all six patients during therapy, but rose to pretreatment values by 8 weeks after cessation of the treatment. In addition, no significant changes were noted in the mean serum HCV RNA levels during and after 24 weeks of ribavirin therapy. Our results indicate that oral ribavirin only transiently lowered serum ALT values and did not efficiently suppress HCV synthesis in patients with chronic hepatitits C infection.     

Hepatitis C virus RNA quantification in right and left lobes of the liver in patients with chronic hepatitis C

Summary. Quantification of hepatitis C virus RNA in liver tissue is likely to be useful in the study of the natural history, pathogenesis, progression and treatment of hepatitis C virus-associated liver disease. Quantitative measurements of hepatitis C virus RNA in liver biopsy samples using the branched DNA (bDNA) signal amplification assay were carried out. The aims of this study were threefold: first, to assess the level of hepatitis C virus RNA in biopsy samples from the right and left lobes of the liver; second, to evaluate the correlation between hepatitis C virus RNA levels in serum and liver; and third, to investigate the relationship between serum and liver hepatitis C virus RNA levels and the severity of hepatic histology in non-cirrhotic patients with chronic hepatitis C. There was a strong correlation ( r = 0.92, P < 0.01) between hepatitis C virus RNA levels in the right and left lobes of the liver as well as a strong correlation between hepatitis C virus RNA levels in liver and serum ( r = 0.82, P < 0.01). However, there was no significant correlation between the severity of hepatic histology and levels of hepatitis C virus RNA in serum and liver among patients with chronic active hepatitis classified according to Knodell's hepatic activity index (KI). Our results indicate that hepatitis C virus RNA quantification from a single liver biopsy is representative of both lobes in patients with chronic hepatitis, and suggest that serum hepatitis C virus RNA levels are a meaningful reflection of hepatitis C virus RNA levels in the liver.