National Institutes of Health
American Liver Foundation

NIH Consensus Development Conference on
Management of Hepatitis C

Diagnostic Tests for Hepatitis C

David Gretch, M.D., Ph.D.

Diagnostic tests for hepatitis C (HCV) can be divided into two general categories: (l) serologic assays which detect anti-HCV antibodies, and (2) molecular assays which detect, quantify, and/or characterize HCV RNA genomes within an infected patient. Serologic assays have been subdivided into screening tests for anti-HCV and supplemental antibody tests. Molecular assays can be divided into qualitative tests for HCV RNA, quantitative tests for assessing viral RNA levels, and HCV genotype tests.

Screening Assays for Anti-HCV

The main screening assay for detecting anti-HCV antibodies is the enzyme immunoassay (EIA). The EIA has many advantages in the diagnostic setting, including ease of use, low variability, ease of automation, and relatively low expense. The first-generation anti-HCV test (EIA- I ) contained a single HCV recombinant antigen derived from the nonstructural (NS) 4 gene, designated clO0-3. (1) Although development of this test represented a dramatic breakthrough in terms of diagnosing HCV infection and reducing HCV transmission via blood transfusion, (2-5) EIA-I lacked optimal sensitivity and specificity and was subsequently replaced in 1992. (6) The EIA-2 test contains HCV antigens from the core and NS3 genes in addition to the NS4 antigen, and thus represents a multi-antigen EIA. (7) Introduction of the new antigens led to a substantial improvement in sensitivity and a slight increase in specificity relative to the EIA- I (Table 1 ) (7-15) The use of core and NS3 antigens in the EIA-2 test shortened the average "window period" for HCV seroconversion by 4-10 weeks relative to the EIA-I test.l¡ l6 A third-generation antiHCV test (ela-3), which contains reconfigured core and NS3 antigens plus an additional HCV antigen (NSS) not present in the EIA-2, (17-20) has recently been approved for screening blood products. While preliminary studies suggest an incremental improvement in sensitivity in blood donors, immunosuppressed populations, and liver clinic populations, the specificity of the EIA-3 test has not been adequately defined in the routine diagnostic setting, and utility of the NS5 antigen in this assay has been controversial.

A progressive improvement in sensitivity of detection of anti-HCV has been accomplished by the three generations of EIA screening assays (Table 1). However, testing in high-prevalence populations has indicated that not all patients with active HCV infection (e.g., HCV RNA positive) are identified with the EIA screening tests. Preliminary studies suggest the envelope 2 (E2) antigen may be a good candidate for subsequent versions of the EIA test. (21-23) Although false-positive EIA testing remains a problem in low-prevalence populations, the accuracy of the EIA-2 test is very good in high-prevalence populations, and therefore, supplemental anti-HCV tests may not be necessary in high-risk patients with a positive anti-HCV screen.

TABLE 1. Sensitivity and Accuracy of Anti-HCV Screening Tests

Low-Prevalence (Blood Donor)High-Prevalence (Liver Clinic)
Test SensitivityAccuracy*TestSensitivityAccuracy*
EIA-195-98%30-50%EIA-160-80%70-85%
EIA-299.7-99.8%50-61%EIA-292-95%88-95%
EIA-399.9+%n.d.EIA-397%n.d.
* Percentage of specimens with a positive anti-HCV screen which are also positive by supplemental anti-HCV test. Modified from references cited in text, and Gretch, et al., unpublished data.

Supplemental Tests for Anti-HCV

Supplemental tests for anti-HCV were developed to help resolve false-positive EIA test results. The prototype supplemental test in the United States is the FDA-licensed second-generation recombinant immunoblot assay (RIBA-2), which contains the same HCV antigens as EIA-2 in an immunoblot format. (7) Results are either positive (two or more positive antigens), indeterminate (one positive antigen), or negative. Interpretation of HCV serology depends on the patient risk status (Table 1). For example, in the low-prevalence blood bank setting, (l) about 40-50 percent of specimens with positive EIA results are false positives (i.e., RIBA-negative), and (2) few RIBA-2 indeterminate results are HCV RNA positive. (11-13,24-28) However, in the high-prevalence setting (e.g., the University of Washington Viral Hepatitis Reference Laboratory), approximately 90 percent of EIA-positive specimens are also positive by RIBA (Gretch, et al., unpublished data). In this setting, about 85 percent of RIBA-positive specimens and 20-50 percent of RIBA core or NS3 indeterminate specimens are positive for HCV RNA by PCR. (28-36) A third-generation supplemental test (RIBA-3) has been introduced in Europe, which appears to be more specific than the RIBA-2 test based on a better correlation with RNA PCR results and a reduced number of RIBA-indeterminate results, (31,37-39) as yet, RIBA-3 has not been approved in the United States.

Qualitative Tests for HCV RNA

Detection of HCV RNA in patient serum by highly sensitive tests such as reverse transcription polymerase chain reaction (RT-PCR) has become an increasingly important tool for confirming the diagnosis of hepatitis C and for assessing the antiviral response to interferon therapy. (40,41) The role of tests for HCV RNA in the diagnosis and management of hepatitis C is discussed in subsequent presentations.

Many variations in the RT-PCR assay have been described in the literature, and standardization of such "home-brew" assays has been difficult, as illustrated by a European survey, where only 16 percent of laboratories scored perfectly on a standardized test panel. (42) Numerous factors contribute to RT-PCR variability, including specimen handling and storage, correct design of amplification primers, variability of biochemical reactions, DNA product contamination, and efficiency of postamplification detection systems. (43-50) It is therefore important to emphasize the need for evidence of rigorous proficiency testing by qualified diagnostic laboratories before HCV RNA testing can be reliably used in patient management. Nonetheless, several excellent home-brew assays with proven clinical utility have been described, (48-56) of which the most sensitive can detect HCV RNA at a level of less than 100 copies per ml of patient serum. In this regard, HCV RNA proficiency testing of laboratories in the U.S. has only recently been initiated by the College of American Pathologists.

Roche Molecular Diagnostics has recently introduced the Amplicor test kit for qualitative HCV RNA detection by the RT-PCR technique. (57,58) The kit is reliable, and has built-in controls for assay sensitivity and specificity. The assay was originally designed to test 50 ul of serum, but was found to be 4-10 fold less sensitive than optimized RT-PCR assays in research reference laboratories. (57) However, modifications of the Amplicor test allow HCV RNA detection at less than 100 HCV RNA copies per ml of serum, with a specificity of 97-99 percent (Gretch, et al., unpublished data).

Quantitative Tests for HCV RNA

In addition to being a valuable research tool, the quantitative assessment of HCV RNA levels in patients before, during, and after therapy has tremendous potential for improving the clinical management of chronic hepatitis C. (59) In a recent study of HCV viremia, it was established that HCV RNA levels are relatively stable (without significant fluctuation) in untreated patients with chronic hepatitis C. (60) These findings are important because, although numerous studies have demonstrated that therapy may reduce HCV RNA levels, the assumption that HCV RNA levels are stable before therapy was unproven. The following paragraph briefly describes current methods for assessing HCV "viral load," while application of HCV RNA testing in monitoring of virologic response to therapy is discussed in subsequent sections.

Two different technologies have been developed to assess HCV RNA levels in patient specimens: target amplification methods such as quantitative PCR (Q-PCR), and signal amplification technologies such as branched DNA assay (bDNA). (61) Quantitative PCR tests have been described by several laboratories, and differ markedly in the reported performance characteristics. (61-71) The only standardized quantitative PCR assay available in kit format is the Roche Monitor assay. Unfortunately, experience with this assay has been limited. The main strength of Q-PCR is high analytical sensitivity, with reports as low as 1,000 RNA copies per ml; on the other hand, major problems include high assay variability, and limited linear range. By comparison, the bDNA test has been extensively evaluated, and appears to be highly standardized, although the sensitivity of bDNA assay is 2-3 logs less than PCR-based methods. (61,72-78) Therefore, PCR testing has been recommended on bDNA-negative specimens. (61,73) It is also important to note that a "genotype bias" is possible for all HCV molecular assays because of the extensive genetic heterogeneity of the virus. For example, the first-generation bDNA test (bDNA 1.0) apparently under-reported HCV RNA levels for a subset of HCV genotypes, (76,77) while the secondgeneration test (bDNA 2.0) appears to be more accurate. (79) Unfortunately, additional refinements may be necessary before standardized tests are licensed for routine use in hepatitis C management.

HCV Genotype Testing

HCV is a remarkably heterogeneous family of viruses, with at least six distinct genotypes and numerous subtypes of HCV identified throughout the world. (80-82) Tests to deterrnine HCV genotype fall into two categories: (I) Screening tests which detect point mutations and (2) confirrnatory tests which evaluate larger segments of HCV genes. Commonly used screening tests include 5'-RFLP analysis, coregene nested PCR, and the LIPA assay. (83-85) Confirrnatory tests include nucleotide sequencing and phylogenetic analysis of the El gene or NSSB gene. (86-88) HCV genotype deterrnination is an important aspect of ongoing clinical trials, since HCV genotype may be an independent predictor of response to therapy, as will be discussed later. However, there is as yet little role for HCV genotyping in the routine clinical setting, since optimal treatment regimens have not been defined for different HCV genotype infections.

References

  1. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-4.
  2. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;321:1494-500.
  3. Esteban JI, Esteban R, Viladomiu L, L'Opez Talavera JC, Gonzalez A, Hemandez JM, Roget M, Vargas V, Genesca J, Buti M, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;2:294-7.
  4. Esteban J, Gonzalez A, Hernandez J, Viladomiu L, Sanchez C, Lopez-Talavera J, Lucea D, Martin-Vega C, Vidal X, Esteban R, Guardia J. Evaluation of antibodies to hepatitis C virus in a study of transfusion associated hepatitis. N Engl J Med 1990;323 :1107-12.
  5. Alter MJ, Hadler SC, Judson FN, Mares A, Alexander WJ, Hu PY, Miller JK, Moyer LA, Fields HA, Bradley DW, et al. Risk factors for acute non-A, non-B hepatitis in the United States and association with hepatitis C virus infection. JAMA 1990;264:2231-5.
  6. Alter H. New kit on the block: evaluation of second-generation assays for detection of antibody to the hepatitis C virus. Hepatology 1992;15:350-3.
  7. Younossi Z, McHutchison J. Serological tests for HCV infection. Viral hepatitis reviews 1996;2:161-73.
  8. Takano S, Nakamuar K, Kawai S, Yokosuka 0, Satomura Y, Omata M. Prospective assessment of donor blood screening for antibody to hepatitis C virus by first-and second-generation assays as a means of preventing posttransfusion hepatitis. Hepatology 1996;23:708-12.
  9. Nakatsuji Y, Matsumoto A, Tanaka E, Ogata H, Kiyosawa K. Detection of chronic hepatitis C virus infection by four diagnostic systems: first-generation and second-generation enyme-linked immunosorbent assay, second-generation recombinant immunoblot assay and nested polymerase chain reaction analysis. Hepatology 1992;16:30¡-5.
  10. Hosein B, Fang C, Popovsky M, Ye J, Zhang M, Wang C. Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein. Proc Natl Acad Sci U S A 1991;88:3647-51.
  11. Kleinman S, Alter H, Busch M, Holland P, Tegtmeier G, Nelles M, Lee S, Page E, Wilbur J, Polito A. Increased detection of hepatitis C virus (HCV)-infected blood donors by a multiple-antigen HCV enzyme immunoassay. Transfusion 1992;32:805-13.
  12. Aach R, Stevens C, Hollinger F, Mosley J, Peterson D, Taylor P, Johnson R, Barbosa L, Nemo G. Hepatitis C virus infection in post-transfusion hepatitis. An analysis with first- and second-generation assays. N Engl J Med 1991;325:1325-9.
  13. McHutchison J, Person J, Govindarajan S, Valinluck B, Gore T, Lee S, Nelles M, Polito A, Chien D, DiNello R, et al. Improved detection of hepatitis C virus antibodies in high-risk populations. Hepatology 1992;15:19-25.
  14. Marcellin P, Martinot-Peignoux M, Boyer N, Pouteau M, Aumont P, Erlinger S, Benhamou J. Secondgeneration (RIBA) test in diagnosis of chronic hepatitis C. Lancet 1991;337:551-2.
  15. Lelie P, Cuypers H, Reesink H, van der Poel C, Winkel 1, Bakker E, van Exel-Oehlers P, Vallari D, Allain J, Mimms L. Pattems of serological markers in transfusion-transmitted hepatitis C virus infection using secondgeneration HCV assays. J Med Virol 1992;37:203-9.
  16. Okamoto H, Tsuda F, Machida A, Munekata E, Akahane Y, Sugai Y, Mashiko K, Mitsui T, Tanaka T, Miyakawa Y, et al. Antibodies against synthetic oligopeptides deduced from the putative core gene for the diagnosis of hepatitis virus infection. Hepatology 1992;15:180-6.
  17. Kao J-H, Lai M-Y, Hwang Y-T, Yang P-M, Chen P-J, Sheu J-C, Wang T-H, Hsu H-C, Chen D-S. Chronic hepatitis C without anti-hepatitis C antibodies by second-generation assay: a clinicopathologic study and demonstration of the usefulness of a third-generation assay. Dig Dis Sci 1996;41:161-5 .
  18. Uyttendaele S, Claeys H, Mertens W, Verhaert H, Vermylen C. Evaluation of third-generation screening and confirmatory assays for HCV antibodies. Vox Sanguinis 1994;66:122-9.
  19. Barrera J, Prancis B, Ercilla G, Nelles M, Achord D, Darner J, Lee S. Improved detection of anti-HCV in posttransfusion hepatitis by a third-generation ELISA. Vox Sanguinis 1995;68:15-8.
  20. Atrah H, Ahmed M. Hepatitis C virus seroconversion by a third-generation ELISA screening test in blood donors. J Clin Pathol 1996;49:254-5.
  21. Yuki N, Hayashi N, Kashahara A, Hagiwara H, Mita E, Ohkawa K, Katayama K, Fusamoto H, Kamada T. Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection. Hepatology 1996;23:947-52.
  22. Lesniewski R, Okasinkski G, Carrick R, Van Sant C, Desai S, Johnson R, Scheffel J, Moore B, Mushahwar 1. Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: a new marker of HCV infection closely associated with viremia. J Med Virol 1995;45:415-22.
  23. Zaaijer HL, Vallari DS, Cunningham M, Lesniewski R, Reesink HW, van der Poel CL, Lelie PN. E2 and NS5: new antigens for detection of hepatitis C virus antibodies. J Med Virol 1994;44:395-7.
  24. van der Poel C, Cuypers H, Reesink H, Weiner A, Quan S, Di Nello R, van Boven J, Winkel I, Mulder-Folkerts D, Exel-Oehlers P, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblotassay. Lancet 1991;337:317-9.
  25. Evans C, Tobler L, Polito A, Stewart J, Chien D, Wilber J, Quan S, Delaney S, Kuo G, Busch M. Comparative evaluations of supplemental hepatitis C virus antibody test systems. Transfusion 1992;32:408-14.
  26. Craxi A, Fiorentino G, Di Marco V, Marino L, Magrin S, Fabriano C, Pagliaro L. Second-generation tests in diagnosis of chronic hepatitis C. Lancet 1991;337:1354.
  27. Alter H, Tegtmeier G, Jett B, Quan S, Shih J, Bayer W, Polito A. The use of a recombinant immunoblot assay in the interpretation of anti-hepatitis C virus reactivity among prospectively followed patients, implicated donors, and random donors. Transfusion 1991;31:771-6.
  28. Busch M, Tobler L, Quan S, Wilber J, Johnson P, Polito A, Steane E, Zola A, Bahl C, Nelles M, et al. A pattern of 5-1-1 and c100-3 only on hepatitis C virus (HCV) recombinant immunoblot assay does not reflect HCV infection in blood donors. Transfusion 1993;33:84-8.
  29. Gretch DR, Lee W, Corey L. Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory. J Clin Microbiol 1992;30:2145-9.
  30. Lok A, Chien D, Choo Q, Chan T, Chiu E, Cheng I, Houghton M, Kuo G. Antibody response to core, envelope and nonstructural hepatitis C virus antigens: comparison of immunocompetent and immunosuppressed patients. Hepatology 1993; 18:497-502.
  31. Pawlotsky J, Bastie A, Pellet C, Remire J, Darthuy F, Wolfe L, Sayada C, Duval J, Dhumeaux D. Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay. J Clin Microbiol 1 996;34:80-3 .
  32. Feucht H, Zollner B, Polywka S, Laufs R. Study on reliability of commercially available hepatitis C virus antibody tests. J Clin Microbiol 1995;33:620-4.
  33. Bresters D, Zaaijer H, Cuypers H, Reesink H, Winkel I, van Exel-Oehlers P, van Drimmelen A, Jansen P, van der Poel C, Lelie P. Recombinant immunoblot assay reaction patterns and hepatitis C virus RNA in blood donors and non-A, non-B hepatitis patients. Transfusion 1993;33:634-8.
  34. Lazizi Y, Elfassi E, Pillot J. Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction. J Clin Microbiol 1992;30:931-4.
  35. Martinot-Peignoux M, Marcellin P, Xu L, Bernuau J, Erlinger S, Benhamou J, Larzul D. Reactivity to c33c antigen as a marker of hepatitis C virus multiplication. J Infect Disease 1992;165:595-6.
  36. Weiner A, Truett M, Rosenblatt J, Han J, Quan S, Polito A, Juo G, Choo Q, Houghton M, Agius C, et al. HCV testing in low-risk population. Lancet 1990;336:695.
  37. Damen M, Zaaijer HL, Cuypers HTM, Vrielink H, van der Poel CL, Reesink HW, Lelie PN. Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus. Transfusion 1995;35:745-9.
  38. Soffredini R, Rumi M-G, Lampertico P, Aroldi A, Tarantino A, Ponticelli C, Colombo M. Increased Detection of Antibody to Hepatitis C Virus in Renal Transplant Patients by Third-Generation Assay. Am J Kidney Dis 1996;28:437 40.
  39. Dow BC, Munro H, Buchanan 1, Follett EAC, Davidson F, Yap PL, Simmonds P. Third-generation recombinant immunoblot assay: comparison of reactivities according to hepatitis C virus genotype. Transfusion 1996;36:547-51.
  40. Alter H. To C or not to C: these are the questions. Blood 1995;85 :1681-95.
  41. Houghton M, Weiner A, Han J, Kuo G, Choo Q. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology 1991;14:381-8.
  42. Zaaijer HL, Cuypers HT, Reesink HW, Winkel IN, Gerken G, Lelie PN. Reliability of polymerase chain reaction for detection of hepatitis C virus. Lancet 1993 ;341:722 4.
  43. Busch M, Wilber J, Johnson P, Tobler L, Evans C. Impact of specimen handling and storage on detection of hepatitis C virus RNA. Transfusion 1992;32:420-5.
  44. Davis GL, Lau JY, Urdea MS, Neuwald PD, Wilber JC, Lindsay K, Perrillo RP, Albrecht J. Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients. Hepatology 1994;19:1337-41.
  45. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237-8.
  46. Wang J, Wang T, Sheu J, Lin S, Lin J, Chen D. Effects of anticoagulants and storage of blood samples on efficacy of the polymerase chain reaction assay for hepatitis C virus. J Clin Microbiol 1992;30:750-3.
  47. Bukh J, Purcell R, Miller R. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc Natl Acad Sci U S A 1992;89:187-91.
  48. Fong T, Charboneau F, Valinluck B, Govindarajan S. The stability of serum hepatitis C viral RNA in various handling and storage conditions. Arch Pathol Lab Med 1993;117:150-1.
  49. Cristiano K, Di Bisceglie A, Hoofnagle J, Feinstone S. Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets. Hepatology 1991;14:51-63.
  50. Castillo 1, Bartolome J, Quiroga JA, Carreno V. Comparison of several PCR procedures for detection of serum HCV-RNA using different regions of the HCV genome. J Virol Methods 1992;38:71-9.
  51. Ulrich P, Romeo J, Lane P, Kelly 1, Daniel L, Vyas G. Detection, semiquantification, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferase. J Clin Invest 1990;86:1609-14.
  52. Lin HJ, Shi N, Mizokami M, Hollinger FB. Polymerase chain reaction assay for hepatitis C virus RNA using a single tube for reverse transcription and serial rounds of amplification with nested primer pairs. J Med Virol 1992;38:220-5.
  53. Gretch D, Wilson J, Carithers Jr R, dela Rosa C, Han J, Corey L. Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR. J Clin Microbiol 1993;3 1 :289-91 .
  54. Hu KQ, Yu CH, Vierling JM. One-step RNA polymerase chain reaction for detection of hepatitis C virus RNA. Hepatology 1993;18:270 4.
  55. Cheung R, Matsui S, Greenberg H. Rapid and sensitive method for detection of hepatitis C virus RNA by using silica particles. J Clin Microbiol 1994;32:2593-7.
  56. Schmidt W, Klinzman D, LaBrecque D, Macfarlane D, Stapleton J. Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV-RNA in plasma and peripheral blood mononuclear cells. J MedVirol 1995;47:153-60.
  57. Nolte FS, Thurmond, C, Fried MW. Pre-clinical evaluation of amplicor hepatitis C virus test for detection of hepatitis C virus RNA. J Clin Microbiol 1995;33:1775-8.
  58. Young K, Archer J, Yokosuka 0, Omata M, Resnick R. Detection of hepatitis C virus RNA by a combined reverse transcription PCR assay: comparison with nested amplification and antibody testing. J Clin Microbiol 1995;33:654-7.
  59. Gretch D, dela Rosa D, Corey L, Carithers R. Assessment of hepatitis C viremia using molecular amplification technologies. Viral Hepatitis Reviews 1996;2:85-96.
  60. Nguyen T, Sedghi-Vaziri A, Wilkes L, Mondala T, Pockros P, Lindsay K, McHutchison J. Fluctuations in viral load (HCV-RNA) are relatively insignificant in untreated patients with chronic HCV infection. J Viral Hepatitis 1996;3:75-8.
  61. Gretch D, dela Rosa C, Carithers R, Willson R, Williams B, Corey L. Assessment of hepatitis C viremia using molecular amplification technologies: Correlations and clinical implications. Ann Intern Med 1995;123:321-9.
  62. Besnard NC, Andre PM. Automated quantitative determination of hepatitis C virus viremia by reverse transcription-PCR. J Clin Microbiol 1994;32:1887-93.
  63. Shindo M, Di BAM, Silver J, Limjoco T, Hoofnagle JH, Feinstone SM. Detection and quantitation of hepatitis C virus RNA in serum using the polymerase chain reaction and a colorimetric enzymatic detection system. J Virol Methods 1994;48:65-72.
  64. Kato N, Yokosuka 0, Hosoda K, Ito Y, Ohto M, Om M. Quantification of hepatitis C virus by competitive reverse transcription-polymerase chain reaction: increase of virus in advanced liver disease. Hepatology 993;18:16.
  65. Hagiwara H, Hayashi N, Mita E, Naito M, Kasahara A, Fusamoto H, Kamada T. Quantitation of hepatitis C virus RNA in serum of asymptomatic blood donors and patients with type C chronic liver disease. Hepatology 993;17:545-50.
  66. Kobayashi Y, Watanabe S, Konishi M, Yokoi M, Kakehashi R, Kaito M, Kondo M, Hayashi Y, Jomori T, Suzuki S. Quantitation and typing of serum hepatitis C virus RNA in patients with chronic hepatitis C treated with interferon-~. Hepatology 1993;18:1319-25.
  67. Goergen B, Jakobs S, Symmons P, Hornes E, Meyer zBKH, Gerken G. Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method. J Hepatol 1994;21:678-82 .
  68. Kaneko S, Murakami S, Unoura M, Kobayashi K. Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction. J Med Viro M992;37:278-82.
  69. Yun Z, Lundeberg J, Johansson B, Hedrum A, Weiland 0, Uhl'en M, Sonnerborg A. Colorimetric detection of competitive PCR products for quantification of hepatitis C viremia. J Virol Methods 1994;47:1-13.
  70. Gretch D, Corey L, Wilson J, dela Rosa C, Willson R, Carithers R, Jr., Busch M, Hart J, Sayers M, Han J. Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction: hightiter viremia correlates with advanced stage of disease. J Infect Dis 1994;169:1219-25.
  71. Simmonds P, Zhang L, Watson H, Rebus S, Ferguson E, Balfe P, Leadbetter G, Yap P, Peutherer J, Ludlam C. Hepatitis C quantification and sequencing in blood products, haemophiliacs and drug users. Lancet 990;336:1469-72.
  72. Chan CY, Lee SD, Hwang SJ, Lu RH, Lu CL, Lo KJ. Quantitative branched DNA assay and genotyping for hepatitis C virus RNA in Chinese patients with acute and chronic hepatitis C . J Infect Dis 1995; 171:443-6.
  73. Bresters D, Cuypers H, Reesink H, Mauser-Bunschoten E, van den Berg H, Schaasberg W, Wilber J, Urdea M, Neuwald P, Lelie P. Comparison of quantitative cDNA-PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophiliac patients participating in a controlled interferon trial. J Med Virol 1994;43:262-8.
  74. Lau JY, Davis GL, Kniffen J, Qian KP, Urdea MS, Chan CS, Mizokami M, Neuwald PD, Wilber JC. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993;341:1501 4.
  75. Martinot-Peignoux M, Marcellin P, Gournay J, Gabriel F, Courtois F, Branger M, Wild A, Erlinger S, Benhamou J. Detection and quantitation of serum HCV-RNA by branched DNA amplification in anti-HCV positive blood donors. J Hepatol 1994;20:676 8.
  76. Toyoda H, Nakano S, Kumada T, Takeda 1, Sugiyama K, Osada T, Kiriyama S, Orito E, Mizokami M. Comparison of serum hepatitis C virus RNA concentration by branched DNA probe assay with competitive reverse transcription polymerase chain reaction as a predictor of response to interferon-a therapy in chronic hepatitis C patients. J Med Virol 1996;48:354-9.
  77. Hayashi J, Yoshimura E, Kishihara Y, Yamaji K, Etoh Y, Ikematsu H, Kashiwagi S. Hepatitis C virus RNA levels determined by branched DNA probe assay correlated wtih levels assessed using competitive PCR. Am J Gastroenterol 1996;91:3 14-8.
  78. Eyster M, Fried M, Di Bisceglie A, Goedert J. Increasing hepatitis C virus RNA levels in hemophiliacs: relationship to human immunodeficiency virus infection and liver disease. Blood 1994;84:1020-3 .
  79. Lau J, Simmonds P, Urdea M. Implications of variations of "conserved" regions of hepatitis C virus genome. Lancet 1995;346:425-6.
  80. Simmonds P. Variability of hepatitis C virus. Hepatology 1995;21:570-82.
  81. Marrone A, Sallie R. Genetic heterogeneity of hepatitis C virus: The clinical significance of genotypes and quasispecies behavior. In: Feitelson Mj Zern M, eds. Clinics in laboratory medicine. Vol 16(2). Philadelphia: WB Saunders 1996:429-49.
  82. Bukh J, Miller R, Purcell R. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin Liver Dis 1995;15:41-63.
  83. Simmonds P, McOmish F, Yap PL, Chan SW, Lin CK, Dusheiko G, Saeed AA, Holmes EC. Sequence variability in the S' non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity. J Gen Virol 1993;74:661-8.
  84. Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, Tanaka T, Sato K, Tsuda F, Miyakawa Y, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992;73:673-9.
  85. Stuyver L, Rossau R, Wyseur A, Duhamel M, Vanderborght B, Van HH, Maertens G. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J Gen Virol 1993 ;74:1093-102.
  86. Stuyver L, Van Arnhem W, Wyseur A, Hernandez F, Delaporte E, Maertens G. Classification of hepatitis C viruses based on phylogenetic analysis of the envelope I and nonstructural SB regions and identification of five additional subtypes. ProcNatlAcad Sci USA 1994;91:10134-8.
  87. Simmonds P, Holmes E, Cha T, Chan S, McOmish F, Irvine B, Beall E, et al. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-S region. J Gen Virol 1993;74:2391-9.
  88. Bukh J, Purcell R, Miller R. At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative El gene of isolates collected worldwide. Proc Natl Acad Sci U S A 1993;90:8234-8.

Home Information Guestbook Email


Conference Proceedings Table of Contents

Hepatitis C Consensus Conference Main Page

American Liver Foundation
1425 Pompton Avenue
Cedar Grove, NJ 07009
1-800-GO LIVER (465-4837)

The American Liver Foundation is a national voluntary health organization dedicated to preventing, treating, and curing hepatitis and other liver and gallbladder diseases through research and education.

Copyright © 1996 The American Liver Foundation