The use of real-time PCR to detect hepatitis C virus RNA in dried blood spots from Brazilian patients infected chronically

Collecting and transporting samples for RNA analysis can be challenging, especially in situations where financial resources are limited. In this study, a quantitative real-time PCR (qPCR) for the analysis of HCV RNA was developed and adapted for use with dried blood spot (DBS) samples. A qPCR for HCV 5′NCR, an internal control and a calibration curve were developed, and the sensitivity, specificity and dynamic range of amplification were evaluated using a panel of viruses. Plasma and DBS samples from 100 patients who had completed four weeks of Peginterferon alfa-2b + Ribavirin treatment were collected (DBS on SS903 collection cards and transported at room temperature). After 24 weeks of treatment, samples were collected from 68 of these patients. Of the 168 samples, 2 yielded false-negative results, and 4 yielded false-positive results (sensitivity was 98%, specificity was 94.3%, positive predictive value was 96.1%, and negative predictive value was 96.9%). Additionally, 2039 DBS samples from 1114 patients currently undergoing treatment for a chronic HCV infection in a clinical trial were tested. Only 10 samples out of the 2039 yielded invalid results warranting re-collection of DBS. The detection of HCV RNA in DBS can be a cost-effective strategy for HCV treatment monitoring, especially in settings where resources are limited.     

Integration of hepatitis B virus DNA in chronically infected patients assessed by Alu‐PCR

Hepatitis B virus (HBV) infection is the main risk factor for hepatocellular carcinoma (HCC) worldwide. Integration of HBV DNA into the human genome has been found in >80% of HBV‐related HCC cases. Some studies have, however, found similar integration patterns in tumorous and nontumorous tissues. Thus, the role of integrations for the development of HCC as well as the rate of integration in different stages of infection remain unclear. The aim of this study was to investigate integrations in patients without HCC, representing different stages of chronic HBV (CHB) infection. Extracted DNA in liver biopsies from 74 patients (one with 2 available biopsies) with CHB infection was analyzed by Alu‐PCR. Amplicons were further analyzed by Sanger sequencing. Integration was detected in 39 biopsies (52%) as an amplicon containing both human and HBV sequences by Alu‐PCR with one primer targeting a region in the HBV genome. Integrations were found in patients representing the different stages of CHB infection. A majority of the HBV sequences were located upstream or downstream of nucleotide position 1820, which previously has been identified as a common breakpoint in the HBV genome in integrated sequences. Approximately 60% of the HBV integrations were found in noncoding regions of the human genome. Integrations of HBV DNA into the human genome is an event frequently found in mild phases of chronic hepatitis.     

Strong correlation between liver and serum levels of hepatitis C virus core antigen and RNA in chronically infected patients

HCV core antigen (Ag) and HCV RNA levels were evaluated in matched liver biopsy samples and sera from 22 patients with hepatitis C infection by using the quantitative Architect HCV Ag immunoassay and a real-time RT-qPCR assay, respectively. The data showed a strong correlation between liver and serum compartments of HCV Ag levels (r = 0.80) and HCV RNA levels (r = 0.87). In summary, the serum HCV Ag and RNA levels reflect the intrahepatic values.     

Biochemical response to interferon of chronic hepatitis in drug-addict patients infected with human immunodeficiency virus

The effects of IFN treatment were retrospectively evaluated for 18 drug-addict patients with symptomatic HIV infection and chronic hepatitis C. Most of the patients were receiving concomitant treatment with zidovudine. Seven out of the 18 patients (39%) stopped IFN after less than three months, most of them for non-compliance. Among the 11 patients who completed a 6-12 month period of IFN treatment, 3 (27%) normalized and maintained normal ALT levels during therapy: for 2 of them the response was sustained after IFN discontinuation. The response to IFN therapy was neither correlated to the CD4+ levels nor to the clinical stage of the HIV infection. Instead, the response seemed to be influenced by pre-therapy ALT levels and liver histology. Tolerance to IFN treatment was good. These data show that IFN may be indicated in the therapy of chronic HCV infection for HIV-positive patients.     

Transient reappearance of serum hepatitis C virus RNA observed by real-time PCR during antiviral therapy with peginterferon and ribavirin in patients with HCV genotype 1b

BackgroundThe “response-guided therapy” based on response of hepatitis C virus (HCV) during antiviral combination therapy with peginterferon and ribavirin is important for patients with HCV genotype 1. However, the sensitivity of previous assays for serum HCV RNA is limited.ObjectivesWe evaluated the changes in serum HCV RNA during the combination therapy using a novel method for measurement based on real-time PCR.Study designChanges in serum HCV RNA during the combination therapy were reanalyzed using TaqMan PCR assay in 144 patients with chronic HCV genotype 1b infection who underwent the therapy under HCV RNA monitoring with the Amplicor Monitor assay. Treatment duration was elongated from 48 weeks to 72 weeks in 17 patients based on the time when serum HCV RNA became negative.ResultsIn 9 of 144 (6.3%) patients, serum HCV RNA transiently appeared again on the TaqMan PCR assay after having previously become negative. At the point of reappearance, the Amplicor Monitor assay gave a negative result in all patients, and no flare of alanine aminotransferase activity was observed. Each of the 9 patients achieved an end-of-treatment response but relapsed after the end of treatment, including 3 patients in whom the treatment duration was elongated to 72 weeks.ConclusionsAttention should be paid to this phenomenon in the antiviral treatment for patients with HCV infection. The transient reappearance of HCV RNA in the serum indicates a high likelihood of relapse, and is likely to be missed without frequent measurements by a sensitive detection method.     

实时定量PCR检测马传染性贫血病毒载量方法的建立和应用

目的建立实时定量PCR检测血浆病毒载量的方法，对马传染性贫血病毒（equine infectious anemia virus，EIAV）强毒株攻毒马和疫苗免疫攻毒马血浆中病毒载量进行了跟踪检测，探讨病毒载量和临床疾病状态的相关性。方法以EIAV强毒株LN40序列为标准，在gag保守区设计1对引物和Taqman探针，用于实时定量PCR扩增，用含扩增目的基因的体外转录RNA作标准品，获得标准曲线，对扩增样品进行准确定量。强毒株LN40直接攻毒，或者疫苗株DLV免疫马6个月后用强毒株LN40进行攻毒，跟踪检测攻毒马及免疫攻毒马血浆中EIAV载量情况。结果反应在10^1～10^9copies／ml之间具有良好的线性关系，反应的检出下限为10copies／ml。强毒攻毒马出现发热并最终死亡，发热期间马血浆中EIAV载量与体温呈正相关，载量最高达10^7copies／ml。免疫攻毒马未出现发热，其血浆EIAV载量的总体水平低于强毒攻毒马，攻毒后3个月低至10copies／ml以下。结论成功建立了实时定量PCR检测血浆EIAV载量的方法，并证实了用实时荧光定量PER检测EIAV病毒载量的方法来监测动物感染状态具有可行性，为EIAV致病机制研究和弱毒疫苗免疫保护机制的研究提供良好的技术平台。     

Detection of hepatitis B virus core mutants by PCR-RFLP in chronically infected patients

HBV chronic infection is an important health problem. The HBV core antigen carries several epitopes for T and B cell recognition and the immune response is crucial for determining the outcome of viral infection. Using PCR-RFLP several point mutations were detected in the HBV core ORF of HBV extracted from the serum of 140 chronically infected patients and 86 samples from another 37 patients followed-up in a longitudinal study. Mutations at position 2248 and 2147 (A3) and at 2038 (M2) were found most frequently. The wild type core genotype was found in about 50% of the samples. PCR-RFLP results were confirmed by direct sequencing of amplified products from HBV DNA present in chronically infected patients. The method is rapid and reliable and may be particularly useful for a rapid detection of viral mutants in a large number of patients.     

Chemiluminescent hybridization assay for hepatitis delta virus RNA in serum.

A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.     

Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy

Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.     

Detection and quantitation of hepatitis C virus RNA in feces of chronically infected individuals

Hepatitis C virus (HCV) RNA was detected and quantified in human fecal specimens with the Roche COBAS AMPLICOR system adapted by us for fecal specimens. HCV RNA could be detected in the feces of four of six (67%) patients chronically infected with HCV, with loads up to about 2.8 x 10(5) copies/ml of feces. The same HCV genotypes were observed in feces and plasma as determined by direct sequencing of the 5' untranslated region.     

Analysis of clinical and virological factors associated with response to alpha interferon therapy in chronic hepatitis C

Interferon alpha (IFN-α) therapy is currently the treatment of choice for chronic hepatitis C (HCV) infection, but it fails to achieve a sustained response in approximately 75% of those treated. The factors which determine whether or not an individual will respond to IFN-α are uncertain, although a number of potentially predictive factors have been proposed. In this study a wide range of clinical, demographic, and virological parameters were evaluated in relation to therapeutic outcome in a group of 30 Italian patients with chronic hepatitis C. All patients received 3 MU leukocyte-derived IFN-α three times a week for 6 months and were then followed prospectively for at least 12 months. 53% of patients responded initially, but a sustained response was observed in only 17%. Responders were found to be significantly younger than nonresponders (45.6 ± 3.1 vs. 55.4 ± 2.7), and less frequently cirrhotic (2/16 vs. 7/14). Sustained responders had a mean pretreatment HCV-RNA titer approximately tenfold lower than that of those who did not have a sustained response, but the difference was not statistically significant. HCV genotype was found to be significantly associated with both initial and sustained response. Patients infected with HCV-2a were more likely to respond (89%) than those who were infected with HCV-1 b (37%), and they were also more likely to sustain that response (33% vs. 6%). Geometric mean titers did not vary significantly between the different genotypes. © 1995 Wiley-Liss, Inc.     

Comparison of real-time PCR assays for monitoring serum hepatitis B virus DNA levels during antiviral therapy

The performance characteristics of the RealART and Molecular Beacons assays were compared with those of the Digene Hybrid Capture II assay (ultrasensitive). The results of the RealART and Digene Hybrid assays were related (r = 0.94; P < 0.001) and diverged by 2 orders of magnitude. The RealART assay can be used to effectively monitor serum hepatitis B virus DNA levels.     

Evaluation of quantitative measurements of hepatitis C virus RNA to predict sustained response to interferon by genotype

Hepatitis C virus (HCV) virus load is one of the most important predictive factors for the outcome of interferon (IFN) therapy. Recent technological advances have allowed a more precise measurement of HCV load. However, the exact cutoff values that could be used to predict the outcome of IFN have not been established for each assay. Five recent quantitative assays were evaluated for the measurement of HCV (Amplicor monitor ver 1.0, Amplicor monitor ver 2.0 (GT), Amplicor monitor ver 2.0 (Cobas), Quantiplex branched DNA amplification (bDNA) ver 2.0 and HCV core protein level by enzyme immunosorbent assay) in 209 consecutive patients with chronic hepatitis C, who received IFN therapy. The results of the two second generation Amplicor monitor tests (GT and Cobas) showed the best correlation (r = 0.930), but the other tests also showed relatively good correlations (r = 0.646-0.925). Each method predicted the effect of IFN with comparable predictive efficacy, ranging from 77.0 to 80.8%. Receiver operating characteristic (ROC) curve analysis showed that Amplicor monitor ver 2.0 and bDNA ver 2.0 are superior in predicting the response in genotype 2a. The best cutoff value for predicting the response to IFN was different by genotype, which should be considered in selecting candidates for IFN treatment.     

Quantification of hepatitis C virus RNA in serum by branched DNA-based signal amplification assays.

The objective of the study was to compare the clinical sensitivity and specificity of versions 1.0 and 2.0 of the branched DNA (bDNA)-based hepatitis C virus (HCV) RNA quantification assay, and also to compare the values yielded by the two versions according to the HCV genotype. Serum samples from 268 patients tested routinely by a non-quantitative HCV RNA PCR assay (group A) were tested with version 2.0 of the bDNA assay. Samples from 342 HCV PCR-positive patients with chronic hepatitis C eligible for interferon treatment (group B) were tested with both version 1.0 and version 2.0 of the bDNA assay. Version 2.0 had a clinical sensitivity of 92% (95% confidence interval (CI): 87-97%) in group A and 89% (86-92%) in group B. In group B, the gain in sensitivity with bDNA 2.0 was 16% relative to bDNA 1.0 (P < 0.001). The log values of the two assays correlated with samples positive by both assays (r = 0.83, P < 0.0001), but the distribution of values was larger in samples containing HCV genotypes 2 and 3. The mean ratio of assay 2.0/assay 1.0 values was 1.69 +/- 1.44 (range: 0.33-13.43). The mean ratio was close to 1 with samples containing genotype 1 or 4, but ranged from 0.33 to more than 5. The mean ratio was close to 3 with samples containing genotype 2 or 3, and ranged from 0.5 to more than 13. HCV RNA levels were significantly lower in samples containing genotype 4 than in those containing other genotypes. Sera from 200 anti-HCV-negative, HCV RNA PCR-negative blood donors (group C), and from 164 anti-HCV-negative patients with symptoms of chronic liver disease (group D) were used to assess the clinical specificity of bDNA 2.0. In addition, samples with an HCV RNA titer between 0.2 (assay cutoff) and 0.5 MEq/ml from a group of 546 patients tested routinely for HCV RNA load by bDNA 2.0 (group E) were retested by bDNA 2.0 and by qualitative PCR. The specificity of bDNA 2.0 was 100% (98-100%) in group C and 99% (97-100%) in group D. Among the 41 samples from group E, 38 were positive by bDNA 2.0 retesting (36 were PCR-positive) and three were negative by bDNA 2.0 retesting (all were PCR-positive). It is concluded that version 2.0 of the bDNA assay is markedly more sensitive than version 1.0 and has a good specificity. In contrast with version 1.0, version 2.0 is not influenced by the HCV genotype. The relationship between values obtained with assays 1.0 and 2.0 on clinical specimens is not linear, indicating that HCV RNA titers cannot reliably be calculated from the results of version 1.0.     

Differential flotation centrifugation study of hepatitis C virus and response to interferon therapy

Hepatitis C virus (HCV) appears to circulate in various forms such as native virion, immune complexes, and nucleocapsids during chronic infections. To determine the association of the physicochemical properties of HCV and its response to interferon therapy in patients with chronic hepatitis C, we examined pretreatment serum samples from 43 patients with HCV RNA who had received interferon therapy, using differential flotation centrifugation in a NaCl solution with a density of 1.063 g/ml. After centrifugation, the ratio of HCV RNA in the top and bottom fractions was determined by the polymerase chain reaction and expressed as T/B. Patients with a sustained response to IFN therapy were found to have higher T/B ratios than transient responders who relapsed after treatment (P < 0.01) and nonresponders (P < 0.01). With regards to HCV genotypes, patients with genotype 1b had higher T/B ratios in the sustained response group than in the nonsustained response group (P = 0.001), but patients with genotype 2 had a similar distribution of T/B among the 3 response groups (not significant). These findings indicate that the physicochemical properties of HCV affect the effectiveness of interferon therapy, particularly in patients with genotype 1b. J. Med. Virol. 52:190–194, 1997. © 1997 Wiley-Liss, Inc.     

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R ² = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.     

Genotyping of hepatitis C virus by Taqman real-time PCR.

BACKGROUND: Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries. OBJECTIVES: To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region. STUDY DESIGN: The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples. RESULTS: Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4. CONCLUSION: The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.