Stability of hepatitis C virus RNA in blood samples by TaqMan real‐time PCR

The storage conditions of blood samples for reliable results are very important in hepatitis C virus (HCV) RNA amplification tests used in routine HCV analyses. According to many studies, storage conditions could affect the RNA stability for HCV RNA detection. We have studied HCV RNA stability in blood samples stored at 4°C. Nineteen blood samples containing different HCV RNA levels were stored at 4°C and they were then analyzed by TaqMAN real‐time PCR method. HCV RNA levels remained almost stable (100%) at least for five weeks at this storage condition. However, among them, the stability period was up to 11 weeks in two of the samples. As with these findings, there was a slightly significant correlation between the positivity time and the beginning HCV RNA levels (r=0.474, P=0.040). We conclude that, blood samples can be stored at 4°C for five weeks without any significant difference in detected HCV RNA level by using TaqMan real‐time PCR. J. Clin. Lab. Anal. 24:134–138, 2010. © 2010 Wiley‐Liss, Inc.     

Hepatitis C virus RNA in serum of blood donors with or without elevated transaminase levels

The pattern of hepatitis C virus (HCV) viremia in blood donors who are positive for antibody to HCV (anti-HCV) according to the level of transaminase activity is unclear. A polymerase chain reaction-based HCV RNA detection method was used to study two clearly defined groups of anti-HCV-positive blood donors with repeatedly normal (n = 27) and elevated (n = 17) alanine aminotransferase (ALT) levels. HCV RNA was detected in only 4 of 27 blood donors with normal ALT values and 15 of 17 with elevated ALT values. These results indicate that anti-HCV- positive blood donors with normal ALT levels constitute a heterogeneous group, as HCV viremia is detectable in only a small proportion of cases. Polymerase chain reaction should be useful in the surveillance of anti-HCV-positive blood donors with normal ALT levels, by identifying those who might benefit from further investigation and treatment.     

Hepatitis C virus among blood donors: follow-up study

BACKGROUND: The exact significance of antibodies to hepatitis C virus (HCV) in blood donors remains unknown. Confirmatory tests of anti-HCV- reactive serum and HCV RNA by polymerase chain reaction (PCR) are used to refute a large proportion of false-positive results. STUDY DESIGN AND METHODS: Ninety-two blood donors who were anti-HCV reactive in a first-generation enzyme-linked immunosorbent assay (ELISA) were reevaluated 10 months later with a second-generation ELISA (ELISA-2) as well as with second-generation recombinant immunoblot assay (RIBA-2) and by PCR. RESULTS: Twenty-five (43.9%) of the 57 ELISA-2-positive donors were confirmed as positive by RIBA-2; of these, 84 percent were HCV RNA positive in PCR. Of the 57 who were still anti-HCV positive, 46 were followed up and tested again in the same manner 2 years after the first screening. At that time, the pattern was little changed: 94 percent of RIBA-2- and PCR-positive donors remained positive. Of RIBA-2- and PCR-positive blood donors, 62 percent had abnormal alanine aminotransferase levels in at least one of the three evaluations. Among the anti-HCV-positive donors confirmed by RIBA-2, 60 percent, versus 12.6 percent in the control group, had a significantly (p < 0.001) more frequent risk factor for HCV infection, due to parenteral exposure to blood. CONCLUSION: These data confirm a good correlation between RIBA-2 reactivity and the detection of HCV RNA in a population of anti-HCV- positive blood donors.     

两种定量测定丙型肝炎病毒核酸的聚合酶链反应方法比较

目的：观察荧光定量聚合酶链反应（PCR）PE5700测定丙型肝炎病毒RNA的准确性。方法：所用临床评价血清盘由36份血清组成,其中包括1个5倍倍比稀释系列（7份）、9份阴性血清和20份阳性血清。使用临床评价血清盘比较2种测定方法的重复性、线性和临床样本符合情况。结果：实时荧光定量方法和全自动PCR－ELISA内标法检测系统的线性回归系数分别为0．9732和0．9995。全自动PCR酶联免疫附（ELISA）内标法检测系统不同含量的样本批内变异系数（log10)均比实时荧光定量方法低。临床样本的检测实时荧光定量方法与全自动PCR－ELISA内标法检测系统的相关系数为0．845,结论：实时荧光定量方法具有良好的线性和重复性,同时与全自动PCR－ELISA内标法检测系统有良好的相关性。     

Hepatitis C virus

Hepatitis C virus is a common blood-borne pathogen that is now declining as a new infection in the population. However, women and men who were infected 2 to 3 decades ago are now developing liver damage. To prevent further damage, treatment with IFN and ribavirin is available. Because of adverse events, this treatment requires close supervision over 6 to 12 months, which is often provided by a clinic nurse in collaboration with the physician and pharmacist. Educational outreach to the public and health care providers may help identify patients earlier and promote screening of high-risk groups.     

Hepatitis C virus vaccine

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a worldwide health problem. It is estimated that still 19,000 cases of new infection per year in USA. It has been difficult to analyze neutralizing immunity against HCV because of the lack of appropriate virus culture system. However, neutralizing antibodies against HCV infection in patient sera were reported using recently developed pseudotype virus and JFH-1 HCV culture systems. A neutralizing antibody induction and a vaccine development are necessary for prophylaxis of HCV infection. Here we summarize the recent progress in basic and clinical research toward the vaccine development for HCV.     

Hepatitis C virus kinetics

The balance of virus production and clearance for untreated patients with chronic hepatitis C virus (HCV) results in a decline of viraemia when initiating active antiviral treatment. During the first phase of interferon-alpha therapy, after a delay of about 8-9 h, the kinetics of the viral load is characterized by a rapid dose-dependent decline. This early response can be observed for almost all patients treated with interferon-alpha. After about 24-48 h, the viral decline enters a second phase of relatively slow exponential decay during the following weeks of therapy. Non-responding patients, however, show constant viraemia or even a rebound during this second phase. The rate of the exponential decline of the viral load in responding patients in this second phase is less sensitive to the dose of interferon-alpha and varies considerably among patients. Furthermore, combination therapy with interferon-alpha plus ribavirin does not significantly improve the initial viral decay, although it may prevent more patients from rebounding. Mathematical modelling of viral dynamics reveals high turnover rates of pre-treatment viral production and clearance, and permits the estimation of in vivo half-lives of a few hours for free HCV virions and of 1-70 days for productively infected cells. Infected cell death rate, which determines the second phase decline slope, is predictive of response to treatment. Current models indicate that the early biphasic viral decline is explained if interferon-alpha partially blocks virion production from infected cells, yet they do not rule out additional antiviral or immunological effects. Therapeutic implications are the advisability of use of frequent (daily) and comparatively high initial doses. In conclusion, kinetic analysis of the viral decay during the first weeks of treatment permits the prediction of response at the end-of-therapy and might help to evaluate new drugs and to optimize therapy.     

Hepatitis virus C infection in children

The prevalence of hepatitis C virus(HCV) infection has been becoming much lower in childhood. Blood transfusion was the principal transmission route of HCV in children. Since the year of 1989, screening of the blood products for HCV antibodies has markedly reduced transfusion-related HCV infection. Mother-to-infant transmission occurs in less than 10% of infants born to HCV-infected mothers. The maternal viral load is the most important factor determining the risk. The natural history and the outcome of HCV infection in childhood dependent on host and viral factors. The rate of progression to chronicity is 60% in post-transfusion infection and 80% in vertical infection. Experience of interferon therapy of chronic hepatitis C in children is limited, with about 50% having a sustained response to the therapy.     

Hepatitis C virus and GB virus C/hepatitis G virus viremia in Swedish blood donors with different alanine aminotransferase levels

BACKGROUND: Hepatitis C virus (HCV) is a known blood-borne hepatotropic virus for which antibody screening of blood donors is universally practiced. The newly identified GB virus C (GBV-C) and its strain variant hepatitis G virus (HGV) are of unknown pathogenic significance, and screening of blood donors for this agent has not yet been implemented. Polymerase chain reaction (PCR) is the most sensitive method for detecting HCV viremia and is the only method presently available for the diagnosis of GBV-C/HGV infection. STUDY DESIGN AND METHODS: RNA extracts of sera from 577 anti-HCV-negative blood donors (393 with elevated alanine aminotransferase [ALT] levels, 184 with normal ALT levels) were tested with nested PCR for HCV and GBV-C/HGV directed at the 5'-noncoding regions of the two viruses. RESULTS: One donor with elevated ALT was HCV PCR positive. This donor was anti-HCV negative when recruited to the study but subsequently developed anti- HCV. Of the 19 donors with GBV-C/HGV viremia in the series as a whole, 16 belonged to the group with elevated ALT levels and 3 to the group with normal ALT levels; the group difference in prevalence was nonsignificant (4.1% [16/393] vs. 1.6% [3/184; p = 0.20]). Phylogenetic analysis showed 16 of the GBV-C/HGV isolates to be classifiable as subtype 2a and three as subtype 2b. At follow-up 3 to 5 years later, 11 of 18 donors were still viremic. CONCLUSION: There was no significant difference in GBV-C/HGV viremia in the group with elevated ALT levels and the group with normal ALT levels. The frequency and subtype distribution in the present series were similar to those in other Western countries.     

临床用血中EB病毒核酸检测结果分析

目的探讨临床用血中EB病毒检测的应用价值,为降低输血感染提供依据。方法采用实时荧光定量PCR方法和酶联免疫吸附法,分别定量检测500例悬浮红细胞及100例新鲜冰冻血浆中EB病毒核酸载量,同时检测核酸阳性样本EB病毒相关抗体。结果检测500例悬浮红细胞样本中,EB-DNA阳性例数44例,阳性率为8.8%;该悬浮红细胞样本分离的血浆阳性40例,阳性率为8.0%;核酸阳性样本EB病毒壳抗原(VCA)IgG、EB病毒核抗原(EBNA1)均为阳性,阳性率100%。VCA抗体(IgA)、EB病毒壳抗原(VCA)IgM、EB病毒早期抗原(EA)IgG均为阴性。100例新鲜冰冻血浆EB-DNA均为阴性。结论临床用血EB病毒携带率较高,检测献血者EB病毒核酸、合理选择血液制品对减少输血感染具有重要意义。