Development of a TaqMan assay for the six major genotypes of hepatitis C virus: comparison with commercial assays

A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and <2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.     

Virological tools to diagnose and monitor hepatitis C virus infection

Approximately 200 million people are chronically infected with hepatitis C virus (HCV). Infection with HCV is curable by therapy, with the current standard treatment based on the combination of pegylated interferon-α and ribavirin. Viral eradication is achieved in approximately half of treated patients. In 2011 a new antiviral treatment based on a triple combination with a protease inhibitor will become available. Virological tools are essential to diagnose HCV infection but they have found their principal application in guiding treatment decisions and assessing the virological responses to therapy. These include the anti-HCV antibody assay, measurements of HCV core antigen and HCV viral load and HCV genotyping. The HCV RNA can be ideally assayed by a real-time assay with a limit of detection of 10–15 IU/mL. Monitoring of viral kinetics during the early phases of antiviral treatment is crucial in making treatment decisions such as early stopping rules and also in optimizing the treatment duration. The HCV genotype should be assessed before the start of treatment because it determines the treatment length and ribavirin dose and also offers prognostic information on treatment outcomes as certain genotypes respond more favourably to treatment.     

Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C

Molecular typing of enteroviruses should ideally focus on regions encoding determinants for neutralization. Mapping of monoclonal neutralizing antibodies has shown the VPI protein, in particular its aminoterminal part, encompassing the B-C loop, to be one major antigenic region. We therefore sequenced 570 nucleotides from the 5'-end of the VP1 region of the genome for all 28 echovirus prototypes, and for 61 clinical isolates representing all different echovirus types. An analysis of 133 sequences, including 39 sequences retrieved from GenBank, classified all echoviruses in enterovirus group B confirming results from sequencing within the VP2 region. The nucleotide and amino acid divergence of VP1 sequences of homotypic strains varied from 7.5-23.0% and from 0.0-5.3%, respectively, when compared to their corresponding prototypes, whereas strains belonging to different serotypes these divergences were 22.1-38.9 % and 4.9-16.4 %, respectively. Despite these minimal overlaps, the VP1 sequence was always more similar to that of the homotypic prototype than to that of any heterotypic strain. For 13 out of 14 echovirus types, where multiple isolates were available, the corresponding VP1 sequences diverged more from those of the prototype than from the other homotypic sequences as a reflection of genetic drift. Because there was a complete concordance between the sequences of the region encoding the VP1 aminoterminus and the serotype (P< 0.00001) sequence analysis of this region might complement typing by neutralization, and classify correctly echovirus isolates that may not be typed conveniently by the antisera in hand.     

Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection

Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 × 10³ copies/μg RNA; range: 0 to 3.6 × 10⁴ copies/μg RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001) but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden. J. Med. Virol. 54:265–270, 1998. © 1998 Wiley-Liss, Inc.     

Lack of clinical significance of variability in the internal ribosome entry site of hepatitis C virus

The extreme 5'-proximal sequence of the hepatitis C virus (HCV) genome including the 5' non-coding region (5'NCR) of 341 nucleotide long and the first 30 nucleotides of the core region is highly conserved among different HCV genotypes. It contains a segment termed Internal Ribosome Entry Site (IRES) that regulates the cap-independent translation of HCV-RNA to polyprotein. Sequence variability in this region has important implications for structural organisation and function of the IRES element and could correlate with HCV RNA concentration or response to antiviral therapy. Fourteen patients (seven women, seven men) with chronic hepatitis C were separated into two groups according to their response to antiviral therapy. Seven of these were sustained responders to treatment by Interferon alpha 2b and Ribavirin and seven were non-responders. After cloning-sequencing, the IRES (nt 21 to 374) appears to be structurally highly conserved. However some variability was found between the different isolates obtained: 209 substitutions with a median of four variants/patients. Comparison of the number of variants present in the viral population of the sustained responders and non-responders patients do not show any difference. Positioning of the mutations on the predicted IRES secondary structure showed that the distribution of the mutations and their apparition frequency were different between the two groups. The translation initiator AUG-4 codon, located in the stem-loop IV, is never modified. Variations observed in the IRES are not a parameter of response to antiviral therapy, but the integrity of this region is a necessary condition to maintain its activity.     

Prevalence and clinical significance of circulating cryoglobulins in HIV-positive patients with and without co-infection with hepatitis C virus

Although hepatitis C virus (HCV) is a recognized cause of circulating cryoglobulins, the role of human immunodeficiency virus (HIV) in the pathogenesis of cryoglobulinemia has not been investigated extensively. To evaluate the prevalence of circulating cryoglobulins and to assess the relationship with clinical and virological parameters, 162 HIV-positive subjects (84 anti-HCV(+)) were tested for cryoglobulins, C3, C4, RF, autoantibodies, HIV-viral titer, and CD4(+) count. Anti-HCV-positive subjects were tested for HCV-RNA, HCV-viral titer, and HCV genotype. All patients were examined for the presence of signs or symptoms of vasculitis and tested for cryoglobulins using a standard biochemical assay. Cryoglobulins were found in 30 (18.5%) cases. Of the 30 positive cases, 29 (96.7%) were anti-HCV-positive and 28 (93.3%) HCV-RNA-positive. The presence of cryoglobulins was significantly associated (P < 0.01) with HCV-RNA positivity (OR = 27), liver cirrhosis (OR = 16), decreased levels of C3 (OR = 8.6), C4 (OR = 13.6), increased levels of IgG and IgM (OR = 6.1 and 7.9, respectively), and RF positivity (OR = 6.3), but was unrelated to CD4(+) cell count, HIV viral load, diagnosis of AIDS, HCV viral load and the presence of autoantibodies. Interestingly, the presence of cryoglobulins was not significantly associated with signs and symptoms commonly associated with cryoglobulinemia. In conclusion, HIV infection does not seem to play a significant role in the production of circulating cryoglobulins, which strongly correlates with HCV co-infection and liver cirrhosis. Typical signs and symptoms of cryoglobulinemia do not correlate with the detection of circulating cryoglobulins in HIV and HCV patients.     

Variability of IgM response in hepatitis C virus infection

The IgM and IgG antibody response to various hepatitis C virus (HCV) antigens was studied in 8 patients who acquired posttransfusion HCV infection. IgM anti-HCV was detectable in only 4 of these patients, coincident with (1 patient) or later than (3 patients) the IgG anti-HCV response. Seven patients had initially decreasing IgG anti-HCV titres, indicating passive transfer of antibodies from donor to recipient. All 8 patients showed active IgG seroconversion, as demonstrated by increasing IgG anti-HCV titres, on average, 75 days after infection. Five years after infection, all patients were still reactive for IgG anti-HCV antibodies and 7 were positive for HCV RNA by the polymerase chain reaction (PCR). Two of these PCR positive patients were also reactive for IgM anti-HCV. It is concluded that the serology of HCV infection does not follow the classical pattern of IgM response preceding detection of IgG. The IgM response may be absent, late, or persistent after HCV infection. The serological diagnosis of recent HCV infection should be based on the polymerase chain reaction or rising IgG titres in at least 2 sequential patient blood samples. © 1993 Wiley-Liss, Inc.     

Genomic characterization of hepatitis C virus isolates from Argentina

Thirty-three Argentinian patients infected with hepatitis C virus (HCV) were studied for viral genotyping. The patients included 10 hemophiliac and 4 polytransfused children and 19 adults: 3 polytransfused, 7 dialyzed and 9 sporadic cases. Core-based genotyping permitted the classification of 31 samples. Genotypes II, I and V were the most frequent: 21 (63.6%), 16 (48.4%) and 10 (30.3%) of the 33 patients, respectively. Only one polytransfused patient carried genotype IV. Genotype II was detected in 7 out of 9 sporadic cases. Thirteen patients (39.3%) were coinfected with two genotypes, and 2 others were coinfected with three genotypes. The remaining 2 samples which could not be typed were characterized following the restriction fragment length polymorphism (RFLP) method, and were classified as type 1. One of these had two consecutive transitional mutations in the 5′ untranslated region (5′ UTR). © 1995 Wiley-Liss, Inc.     

Prediction of the efficacy of antiviral therapy for hepatitis C virus infection by an ultrasensitive RT-PCR assay

The efficacy of interferon therapy for hepatitis C virus (HCV) infection improved remarkably. However, virologic relapse occurs in a substantial proportion of patients with virologic response (defined as an HCV RNA level below 50 IU/ml at the end-of-treatment). A highly sensitive RT-nested PCR assay capable of detecting almost a single copy of HCV RNA and a real-time RT-PCR assay to quantify HCV RNA down to 120 copies per ml were developed. The RT-nested PCR assay showed that 1 IU of HCV RNA is equivalent to 12.2 copies. For 28 patients with virologic response (12 relapsers and 16 sustained virologic responders), week-4 and end-of-treatment plasma samples were retested. At week 4, HCV RNA was detected by the RT-nested PCR and qualitative COBAS Amplicor HCV version 2.0 in 8/9 (89%) and 6/9 (67%) samples from relapsers, and in 4/16 (25%) and 2/16 (13%) samples from sustained virologic responders, respectively. End-of-treatment samples with HCV-negative by the qualitative COBAS Amplicor were positive by the present assay in 4/12 (25%) of relapsing patients and 0/16 (0%) of sustained virologic responders. The viral levels detected by the present assay in the Amplicor-negative samples were 3.5-17.3 copies/ml, which is below the detection limit of COBAS Amplicor. In conclusion, the highly sensitive RT-nested PCR assay can predict sustained virologic response at week 4 and virologic relapse at the end-of-treatment more accurately than COBAS Amplicor, suggesting its usefulness in monitoring antiviral therapy for HCV infection.     

Genotypes and titers of hepatitis C virus for predicting response to interferon in patients with chronic hepatitis C

Interferon induces remission in about 50% of patients with chronic hepatitis C, but it is difficult to predict which patients will respond. Host and viral factors were evaluated for correlation with response to interferon in patients with chronic hepatitis C. Recombinant interferon alpha-2b with a total dose of 480-560 million units was given to 136 patients, of whom 74 (54%) responded. Genotypes of hepatitis C virus (HCV) in sera, I, II, III, IV, and V, were determined by poly-merase chain reaction (PCR) with type-specific primers. In 72 patients, pretreatment levels of HCV RNA were titrated by PCR in serial tenfold dilutions of RNA extracted from serum. Response to interferon occurred in 34 (40%) of 85 patients infected with HCV of genotype II, less frequently than in 22 (85%) of 26 with genotype III (P < 0.001) or in 7 (70%) of 10 with genotype IV. Of 51 patients with genotype II HCV, 6 of 8 (75%) with HCV RNA titers <10⁶ responded, more frequently than 4 of 43 (9%) with titers ≥ 10⁶ (P < 0.001). Responders were younger than non-responders (45.7 ± 11.7 vs. 50.3 ± 9.6 yr) and had received transfusions less frequently (26/74 or 35% vs. 37/62 or 60%, P < 0.01). Response to interferon correlated inversely with the severity of liver histopathology. These results indicate that response to interferon is influenced by HCV genotypes and pretreatment levels of HCV RNA in serum. © 1994 Wiley-Liss, Inc.     

Strategies for reliable diagnosis of hepatitis C infection: the need for a serological confirmatory assay.

The aim of the study was to examine whether the diagnosis of Hepatitis C (HCV) infection can be obtained reliably without using an immunoblot-based confirmation assay. 1,708 EIA-reactive serum samples were examined retrospectively for (i) optical density value in the screening assay, (ii) reactivity in an immunoblot assay, and (iii) result by RT PCR. In 1,394 (81.0%) samples positive results were obtained by both the HCV EIA and the confirmation assay. OD-values > or = 2.2 were observed in 1026 of these samples, but covered the range from 0.4 to 2.1 in the other 368 samples. The combination of HCV EIA reactivity and indeterminate immunoblot assay was observed in 134 (7.8%) serum samples. HCV RNA was detected in 58 cases by PCR. The OD-values of these 58 samples ranged from 0.4 to >2.2. Especially reactivity against the core recombinant protein was indicative of PCR positivity. The reactivity by the HCV EIA could not be confirmed by immunoblot assay or PCR in 180 (10.5%) sera. These false reactive sera showed OD values by EIA from 0.3 to 2.1. It is concluded that no threshold values can be defined which would allow differentiation between positive, indeterminate, and false reactive result by HCV EIA without producing an unacceptably high number of false negative diagnoses. Not using immunoblot-based confirmation would result in many additional PCR examinations. Therefore, confirmation of reactive HCV EIA results by a serological confirmatory assay must remain an essential part of the diagnostic procedure.     

Mother-to-infant transmission of hepatitis C virus: rate of infection and assessment of viral load and IgM anti-HCV as risk factors

One hundred twenty-six mother-infant couples were studied and 105 exposed babies were monitored for at least 12 months to define the risk of mother-to-infant HCV transmission. Infection occurred in 5 out of 76 infants (6.6%) born to 69 viraemic mothers and in none of 29 born to 26 non-viraemic mothers. Only one child was HCV RNA positive one month after birth, while the remaining children became positive at the 3rd to 4th month. HCV genotypes of the babies matched those of their mothers. No difference was found between women who transmitted the virus and those who did not with regard to age, history of drug abuse, HIV infection, ALT abnormal values, HCV genotype, type of delivery, and breast-feeding. Four out of 5 infected infants were born to mothers with IgM anti-HCV (P = 0.04). The mean viral titre in transmitting women (10(7.2)) was higher than in non-transmitting (10(6.5)), and the proportion of mothers with viral load > or = 10(7) was statistically higher in transmitting than non-transmitting women (P = 0.03). These data show that HCV perinatal infection is a rare event and suggest that IgM positivity and high viral load (> or = 10(7)) in the mother are independent variables correlated with HCV transmission (O.R. = 14.5; 95% CI: 1.3-160.7 and O.R. = 16.3; 95% CI: 1.5-179.9, respectively).     

Immunoglobulin G antibody avidity assay for serodiagnosis of hepatitis C virus infection

It has been reported that the avidity of specific IgG antibody is lower in primary viral infection than in chronic viral infection. However, few studies have been reported on the IgG avidity in hepatitis C virus (HCV) infection. In the present study, 36 patients with antibody to HCV (anti-HCV) were examined for IgG avidity by an enzyme immunoassay with or without urea elution. The avidity index was significantly low in patients with primary HCV infection (7.7 +/- 6.8%, mean +/- SD), compared with patients with chronic HCV infection (77.0 +/- 21.8%) and individuals with past HCV infection (44.5 +/- 12.6%). Temporal changes of IgG avidity were examined in six patients with primary HCV infection. The avidity index was low in the acute phase of the infection and then increased with time. These results suggest that the avidity assay for IgG anti-HCV is a useful method for distinguishing primary HCV infection from chronic or past HCV infection.     

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.     

Rapid detection of hepatitis C virus RNA by direct capture from blood

A new diagnostic assay for hepatitis C virus RNA detection is described. HCV genomic RNA is captured onto streptavidin-coated magnetic beads by solution hybridization with biotinylated complementary oligonucleotides. The specificity of the capture assay is confirmed using different capture oligonucleotides as well as sera representing different types of HCV. Sensitivity was determined by testing serial dilutions of a HCV infected plasma. A panel of 50 sera was tested for anti-HCV by a Line Immunoassay and for HCV-RNA by both a conventional guanidinium extraction method and the new capture assay. The specificity of the capture assay was 95.8% and the sensitivity was 92.3% compared to the standard protocol. This method provides a rapid and simple alternative for HCV-RNA detection in blood samples. © 1994 Wiiey-Liss, Inc.     

Evaluation of two simplified methods for genotyping hepatitis C virus

A number of different approaches have been used for genotyping hepatitis C virus (HCV). Two simplified methods were evaluated, both of which used polymerase chain reaction (PCR) to amplify products from the 5′ non-coding region of HCV: non-isotopic restriction fragment length polymorphism (RFLP) analysis and type-specific PCR. Sixty-four viraemic patients suffering from chronic HCV infection were studied using these two techniques; 25/64 samples were further tested with a commercial serotyping ELISA based on synthetic NS4 antigen (Murex, U.K.). The results of the three typing methods were generally in agreement with each other. When only the predominant genotype identified by each method was analysed, the 3 methods had 100% agreement. RFLP did not detect any mixed infections and it was unsuccessful in 16/64 (25%) samples. Both type-specific PCR and serotyping ELISA detected mixed infections. However, serotyping ELISA did not give typeable results in 7/25 (28%) samples, whereas type-specific PCR gave typeable results in all 64 samples. Type-specific PCR detected more mixed infections than serotyping ELISA. Direct sequencing of four PCR products with indeterminate RFLP confirmed changes in restriction enzyme recognition sites. The sequences also confirmed the validity of the predominant genotype in cases of apparent mixed reactions. It is possible that some of these cases were artefacts as a result of quasispecies. J. Med. Virol. 52:35–41, 1997. © 1997 Wiley-Liss, Inc.     

Serological and molecular screening for viruses in blood donors from Ntcheu, Malawi: high prevalence of HIV-1 subtype C and of markers of hepatitis B and C viruses

Chronic hepatitis C in children is characterized by milder forms of liver damage than those found in adults. Such a difference has been attributed to a low viral load in children that may lead to poor recognition of infected cells by the immune system. One approach that could be used to confirm this hypothesis may be to examine the number of infected hepatocytes in liver biopsies. Paraffin embedded liver biopsies from 21 children and 15 adults with chronic hepatitis C virus (HCV) infection (with a similar duration of the infection) were hybridized in situ and the percentage of infected hepatocytes was correlated with the histological activity index, alanine aminotransferase levels and HCV viraemia levels. Histological activity index and HCV viraemia levels were statistically higher (P < 0.05 and P < 0.01 respectively) in adults than in children, and the percentage of infected hepatocytes was higher in adults (11.0 +/- 19.7%) than in children (4.6 +/- 3.6%), although it did not reach statistical significance. Also, the percentage of infected hepatocytes correlated with HCV-RNA concentration in serum in both children (r = 0.683, P = 0.001) and adults (r = 0.768, P = 0.001). The results show that liver damage in children with chronic hepatitis C is not related to the extent of infection in the liver. This findings support the hypothesis of that liver injury in chronic HCV infection is mediated by the host immune response.