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The status of hepatitis C virus screening
The Status of Hepatitis C Virus Screening Jean-Pierre Allain
EPATITIS C VIRUS (HCV), the main etiologic agent of the clinical entity formerly known as non-A, non-B hepatitis, was discovered in 1989 by a group of collaborating scientists from Chiron and the Centers for Disease Control. 1 Recombinant proteins were produced and used for the detection of antibodies to the new agent. 2 This assay has proven to be extremely valuable for the diagnosis of HCV infection. Because non-A, non-B hepatitis was by far the most common transfusion transmitted infection, the newly developed test was applied to the screening of blood donors. Subsequently, significant improvements were made in test performance to achieve the demanding requirements of blood donor screening. During the past 7 years, a continuous effort has been made by Chiron, its licensees, and research units to improve test performance both in sensitivity and specificity, as had occurred previously with antibody screening assays for human immunodeficiency virus (HIV) or human T cell lymphotropic virus H I . However, access to the HCV patent has been restricted to a limited number of diagnostic companies. The usual competition for quality and price from which the public benefits did not take place to the full extent that would be desirable. Very early on, it was recognized that antibody to HCV was more difficult to detect and its clinical significance more difficult to interpret than other viral markers such as antibodies to HIV. In the absence of an HCV antigen assay, the recent availability of genomic amplification techniques has provided the tools to complement antibody screening. This review summarizes the progress made during the relatively short history of HCV antibody screening and confirmation tests as well as the critical role played by genomic amplification in the interpretation of screening test results. The extent of these difficulties might lead, in a not too distant future, to a genomic screening test for
H
From the Division of Transfusion Medicine, Department of Haematology, University of Cambridge, Cambridge, UK. Address reprint requests to Professor Jean-Pierre Allain, MD, PhD, Division of Transfusion Medicine, East Anglia Blood Centre, Long Road, Cambridge, CB2 2PT, United Kingdom. Copyright 9 1998 by W.B. Saunders Company 0887-7963/98/1201-000553.00/0
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HCV to virtually eliminate the risk of HCV transmission by blood product transfusion. THE HISTORY OF HEPATITIS C VIRUS ANTIBODY SCREENING
First Generation Screening Tests The isolation by immunoscreening of a small fragment of the HCV nonstructural protein 4 (NS4) led to the development of a prototype radioimmunoassay (RIA) based on the recombinant 5-1-1 protein. 2 The high proportion of clinical and posttransfusion non-A, non-B hepatitis cases reacting with protein 5-1-1 provided strong evidence that these antibodies were a specific marker of this elusive hepatitis viral agent. Subsequently, a solid body of data became available clearly showing the presence of antibodies to 5-1-1 in many cases of non-A, non-B hepatitis. 3,4 A larger and more effective recombinant antigen called cl00 was then used in an RIA that showed that antibodies to the putative viral agent were present both in donors and in recipients implicated in post-transfusion non-A, non-B hepatitis. In addition, an American population of blood donor showed a rate of positivity ranging from 0.5% to 8%. 5 Antibody to 5-1-1 was correlated with both alanine amino transferase levels and the presence of anti-HBc that had previously been associated with non-A, non-B post-transfusion hepatitis. Very quickly, insufficiencies in the performance of the cl00-based enzyme-linked immunosorbent assay (EIA) ~ was pointed out by many investigators. 6-8 Sensitivity was insufficient as a large proportion of clinically defined non-A, non-B hepatitis were anti-cl00 negative, and antibody was often detectable only weeks after the clinical development of hepatitis. 3 Specificity was impaired by a variety of factors. 9q2 The awaited development of assays using the recombinant 5-1-1 and cl00 antigens coated on a strip of nitrocellulose to capture antibodies detected in an enzyme immunoblot format provided the first means of confirmation. 13,14 Other manufacturers used confirmatory blocking or synthetic peptide assays. 15 Although the vast majority of clinically selected individuals with non-A, non-B hepatitis, or those at high risk of blood borne viral infection such as hemophiliacs or Transfusion Medicine Reviews, Vo112, No 1 (January), 1998: pp 46-55
HEPATITIS C VIRUS
47
tially higher rate of seropositivity was found in high-risk groups, such as hemophiliacs. 24-26 In blood donors who were positive with the firstgeneration assay, 40% to 60% repeatedly reactive samples did not react with the second-generation assays; these negative samples could not be confirmed. 27-29 In untested blood donors, secondgeneration screening assays based on recombinant proteins showed higher sensitivity and specificity.3~ In the United Kingdom, EIA positivity rate ranged from 0.5% to 1% and from 0.2% to 0.4% with firstand second-generation tests, respectively.31 However, the specificity of second-generation screening assays was still far from optimal because nearly 50% of reactive samples could not be confirmed. To overcome this difficulty, several manufacturers attempted to use screening assays that included synthetic peptides from highly immunogenic portions of the core, NS3, NS4, and NS5 regions. They considerably improved specificity but lacked sensitivity.3~ These features were not surprising as synthetic peptides do not contain the potentially reactive impurities from the bacteria used to produce recombinant proteins but have a restricted spectrum of epitopes derived from short segments of a model virus, not reflecting the considerable genetic diversity of HCV.
intravenous drug abusers, were confirmed positive, l some false-positive results were obtained) 6 Nineteen percent to 33% seropositive blood donors were confirmed, indicating a high rate of false positivity. 15,17,~8The test's lack of specificity and relatively poor clinical correlation were taken as sufficient arguments to postpone the screening of blood donors in some countries.~9.2~
Second-Generation Screening Tests A second generation of anti-HCV EIA screening was developed and used in 1991. In some assays, recombinant HCV proteins, particularly from the putative structural antigens c-22 core and nonstructural c-33 from the NS3 region, were added to the NS4 cl00 used in the first-generation assays. In other assays, synthetic peptides derived from the immunogenic sequences of the core, NS3 and NS4 from the prototype virus were used. 2~ As shown in Table 1, five tests became available, two of them totally, or partially, based on synthetic peptides. The introduction of these new antigens nearly doubled the anti-HCV positive rate and reduced the window period by several weeks in patients with acute non-A, non-B hepatitis, explaining many of the antibody/clinical discrepancies observed with the first-generation screening assays. 22,23A substan-
Table 1. HCV Antibody Screening and Confirmatory Assays Recombinant Antigens Core
NS3
Assay Ortho 1st generation EIA Abbott 1st generation EIA Ortho 2d generation EIA Abbott 2d generation EIA Murex EIA UBI-EIA SANOFI EIA Ortho 3d generation EIA Abbott 3d generation EIA RIBA.1 RIBA.2 RIBA.3 INNO-LIAt Murex WB Dediscan (Sanofi)
NS4 c100
+ + +
Synthetic Peptides
NS5 5-1-1
Core
E2/NS1
+ + + +
+ + +
+ c200" c200
+
+ +
+
+ +
+ +
NS5
+ +
+
+ + +
+ +
NS4
+ + • 4
+
+ +
+ +
+
Note. c100, 5-1-1 are designations of recombinant proteins initially used by Chiron Corporation. *c200 is a fusion protein of the NS3 and NS4 recombinant proteins. tlNNO-LIA from Innogenetics uses four separate core peptides of HCV. Other Abbreviations Used: NS3, nonstructural protein 3; NS4, nonstructural protein 4; NS5, nonstructural protein 5; E2, envelope 2; WB, Western blot; RIBA, recombinant immuno-blot assay; EIA, enzyme-linked immunosorbant assays; UBI-EIA, United Biochemical Incorporation.
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Third-Generation Screening Tests In 1993, a third generation of anti-HCV screening assays was introduced (Table 1). The main feature was the addition of a recombinant portion of the NS5 region of the HCV polyprotein. In some assays, the NS3 and the NS4 antigens were presented as a fusion protein c200. These changes did not significantly improve the sensitivity of antiHCV detection, but most of the added sensitivity was related to an improvement in anti-NS3 detection rather than a n t i - N S 5 . 32-34 In contrast, the addition of the NS5 antigen reduced the specificity obtained with the second-generation EIA. 35,36Most additional reactive samples that reacted only with the NS5 protein on the immunoblot confirmatory assay did not contain HCV RNA, suggesting falsepositive reactions. 36,37 For these reasons, secondgeneration assays were considered to be of superior performance and prefered to third-generation tests by many investigators.
Other Immunoglobulin G Detection Assays Commercial HCV antibody screening assays are all based on recombinant proteins from core, NS3, NS4, or NS5 proteins of various length, produced in various expression systems. Some also use core peptides coated on microtitre plates or polystyrene beads. The test format, as in screening assays for other viral markers, is uniformly an indirect antiglobulin EIA. As early as 1991, the high immunogenicity and high level of sequence conservation of the N-terminus of the core protein of HCV led to the development of core only or core plus other synthetic peptide-based EIAs. 21,38;39 Data collected indicated that HCV was relatively poorly immunogenic, antibody titres being often low and to some extent parallel to the level of viremia. 4~ In infected individuals, HCV elicited an immunoglobulin G (IgG) immune response to the various antigens over variable and often prolonged periods. In some individuals, antibodies, often to NS4, but also on occasion to core or NS3, did not occur or reach detectable levels. 42 In addition, HCV has a high level of genomic and antigenic variability that may be reflected in antibody detection to be used for serologic genotyping. 43 These peculiar features of HCV infection explain the relative failure of screening assays based on short peptide sequences covering a limited number of epitopes derived from a single (not always the most prevalent) viral sequence.
JEAN-PIERRE ALLAIN
Unlike the HIV antibody assays, the HCV envelope proteins are remarkably absent from the screening panel of antigens. The E2 glycoprotein has been the focus of considerable interest both because of its high level of antigenicity and its high variability in its N-terminus portion where hypervariable regions 1 and 2 are located (HVR1 and 2). E2 has been expressed in a fully glycosylated form in mammalian cells 44 and used in antibody assays. 45,46 Similar to other HCV recombinant antigens, a high (->90% in HCV RNA-positive patients) frequency of reactivity was observed. The low level of expression and higher cost of this antigen probably explain its absence from the screening assays. Sequence analysis and studies of specific E2 peptide reactivity indicated a small number of cross-reactive epitopes outside of the HVR regions and a largely restricted reactivity of the HVR highly immunogenic epitopes. 47-5~ The discrepancy observed between the high prevalence of anti-recombinant E2 and the low prevalence of anti-E2 peptides suggest the importance of conformational and/or carbohydrate-related epitopes.
lgM and IgA Anti-Hepatitis C Virus Estimations IgM anti-core peptides of HCV have been tested for in patients with HCV infection and donor screening with 23% to 86% of patients with chronic HCV-related liver disease found to be positive for IgM anti-HCV, 51,52and 33% in asymptomatic HCV carriers. 4~ Screening blood donors for IgM anti-HCV yielded a prevalence three times lower than that for IgG anti-HCV; although no confirmation assay for IgM is available. IgA antibody to HCV core peptide could not be detected in one study of patients with chronic liver disease. 51 CONFIRMATION OF HEPATITIS C VIRUS ANTIBODY SCREENING
As observed with other antibody assays, the positive predictive value (PPV) for the diagnosis of past or present infection decreased sharply with the prevalence of true infection in the studied population. PPV is particularly low in populations of healthy people at low risk of infection such as blood donors. Supplementary tests are necessary to differentiate between true anti-HCV antibodies and false positivity. To be effective, confirmatory assays should differ from the screening assay not only in the test format but also the antigens used for
HEPATITIS C VIRUS
detection. In the case of HCV antibody, these basic rules have been only partially followed, creating some difficulty in interpretation of test results. The first anti-HCV confirmatory assay and its subsequent modifications were called recombinant immunoblot assays (RIBA). Antigens used in the first generation (RIBA.1) were 5-1-1- and cl00coated on a strip of nitrocellulose together with an IgG procedural control and SOD as the main source of false positivity. Reactivity to both antigens was required for positivity. Similarly to the firstgeneration screening assays, the sensitivity and specificity of RIBA.1 were low and the disadvantages of using the same antigens as the screening assay in the same indirect EIA format were noted. 54-57 cl00 expressed in Eschericheria coli instead of yeast and synthetic peptides derived from the cl00 sequence were used by manufacturers to improve specificity.55,s8 Together with the second-generation screening assays, RIBA.1 was soon replaced by RIBA.2. Three screening antigens (c22, c33, and cl00) and 5-1-1 were coated on the nitrocellulose strip. Confirmation was defined as the reactivity of at least two of these four HCV antigens, although many investigators considered 5-1-1 and cl00 reactivity as insufficient to define confirmation because essentially they were overlapping epitopes. 54.55 Nonetheless, sensitivity and diagnostic value improved considerably.23,56 When applied to a population of blood donors positive with firstgeneration screening assay, RIBA.2 increased the confirmation rate slightly and many indeterminate results became negative. 55,57In patients chronically infected by HCV (HCV RNA-positive), RIBA.2 reacted with three antigens in 60%, two antigens (c22 and c33) in 25% to 30%, and with only one antigen (either c22 or c33) in 6% to 10% of samples .58 In second-generation screening test positive blood donors, 50% were RIBA.2 negative, 25% confirmed, and 25% indeterminate. 59,6~ Fifty percent to 70% of indeterminate samples were reacting with the c22 (core) antigen, the rest nearly equally distributed between c33 and cl00 reactivity.59,6~,62The significance of indeterminate RIBA.2 results were variably interpreted: false positivity, residual antibody postrecovery of HCV infection, partial antibody pattern related to HCV subtypes, 63 or true persistent infection confirmed by the presence of HCV RNA. The general opinion was that most were false-positive because they
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were either not reacting with other screening ass a y s , 59,60,64,65 o r with other confirmatory assays. 15'60'66-68 In addition, they were often nonreproducible over time, 62 were negative for HCV RNA, 59-61 and were not associated with clinical or histologic evidence of liver disease. 69 However, 10% to 25% of c22 indeterminate samples contained HCV RNA, suggesting that some individuals generate detectable antibodies only to this antigen, either because of a low level of viral replication7~ or because of a different HCV subtype. 63 This situation is more frequent in patients receiving immunosuppressive treatment. 68 HCV RNA has seldom been found in anti-c33 and exceptionally in anti-cl00 indeterminate samples.S4,58,65,71 The relative lack of specificity of RIBA.2 was considerably reduced with RIBA.3, which detected antibody to HCV core by specific peptides. 37,72-74 Sensitivity was also improved by a better detection of anti-NS3 antibodies. 73,74 Many HCV RNAnegative samples reacted exclusively with the NS5 antigen revealing that false-positive screening tests were often related to this antigen. As with cl00, virtually no anti-NS5 indeterminate samples were found concomitantly HCV RNA-positive. 75 Although RIBA.3 yielded approximately 10% indeterminate results, 76 some false-positive results were still obtained, as they were not detected by alternative confirmatory assays except when HCV RNApositive. 72 Indeterminate results are not the only difficulty in the interpretation of RIBA or other confirmatory assays. A few cases of blood donors reacting with two antigens such as c33 or NS5 or NS4 but with no other epidemiological or clinical evidence of HCV infection have been described. 77-8~Analysis of such data suggest that such confirmed antibody results are, in fact, false-positive. Reasons for these observations are unclear. 81 These indeterminate test results clearly indicate that, contrary to that found with HIV serology, HCV screening and confirmatory data are in some cases insufficient to provide reliable information for a correct diagnosis and appropriate patient counselling. The simultaneous development of HCV RNA detection methods by genomic amplification provides critical additional information. The classical confirmatory scheme, which includes initial screening assay, repeat screening assay in duplicate, confirmatory assay of repeatedly
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JEAN-PIERRE AL/AIN
reactive samples, has been challenged on the basis of higher efficacy and improved cost-effectiveness by the following algorithm: initial screening EIA-1, repeat screening EIA-1, repeat screening EIA-2, provided the second screening assay uses different antigens than the first o n e . 39,82,83
HEPATITIS C VIRUS RNA DETECTION AND CONFIRMATION
HCV is a persistent virus in most infected individuals. 3 Approximately 10% of individuals seem to recover from the infection as HCV is not detected in serum or liver and both humoral and cellular immunity is present in the host. However, protection is fragile and superinfecfion with another viral strain or possibly the same viral strain can occur naturally or experimentally. 84,85 During the window period, circulating viral genome is the only detectable marker. There is also evidence that HCV can chronically circulate without detectable antibody. HCV transmission by transfusion of antibody positive units is correlated with the presence of HCV RNA detected by sensitive genomic amplification methods, s6-8s HCV RNA detection has a critical role to play in the diagnosis of HCV infection and the confirmation process of positive screening results, particularly in a low prevalence population. HCV genomic detection can be done by direct hybridization (Chiron branched DNA, bDNA) or nucleic acid amplification using the polymerase chain reaction (PCR). bDNA is quantitative but relatively insensitive (1 • 105 genome equivalents/ mL) and inadequate for the purpose of confirmation. Amplification methods such as PCR are applied to cDNA after nucleic acid extraction and reverse transcription. It can be performed with either single amplification step or in a nested format which requires two primer pairs and increases the risk of contamination. 8996A sensitivity below 1,000 genome equivalent/mL can be achieved, which seems sufficient for clinical purposes, and kits are commercially available. 97-99In high- and low-risk groups, symptomatic or asymptomafic, with normal or increased transaminases level, 80% to 95% of confirmed HCV antibodypositive individuals are HCV carriers. Viremia ranges from 1 • 102 to 1 • 107/mL of blood with large fluctuations seen over time in the same patient, but most patients are consistently vire-
mic. 25,96,100 This very high prevalence of HCV
viraemia is useful in the confirmation process. From a clinical point of view, HCV RNA is the highest single predictive marker of HCV infection. HCV RNA positivity has been shown to be highly predictive of infectivity by transfusion. 101,102 For confirmatory purposes, HCV genomic amplification methods, although commercially available, present technical difficulties and are associated with the risk of false-positive results, unless performed in a suitable environment including the physical separation of reagent, sample preparation, and amplification steps. False-negative results can also be obtained when endogenous inhibitors are present, when anticoagulants like heparin are used to collect the samples. It is recommended that tests be performed in reference laboratories using experienced personnel. 1~176 Genomic amplification is necessary to the confirmation process in a limited number of situations, such as an indeterminate confirmatory result or positivity with only two reactive antigens. However, for the purpose of counselling, the absence of viremia suggests strongly a low or no risk of transmission and high probability of the individual being amongst the 10% who recover from HCV infection. CLINICAL RELEVANCE OF ANTI-HEPATITIS C VIRUS SCREENING
Screening for HCV antibody has provided the means to diagnose approximately 85% of non-A, non-B hepatitis during the postacute and chronic phases. The third-generation assays in current use do not detect HCV during the window period that can last for several weeks, and rare cases of antibody-negative chronic HCV carders. Donors in these situations constitute the source of residual post-transfusion infections. Blood donor anti-HCV screening has reduced dramatically the incidence of post-transfusion non-A, non-B hepatitis, l~176 Antibody detection might still be improved with the use of an assay with higher sensitivity to include individuals with low viremia who produce very low levels of antibody: This objective could be reached by replacing the NS5 antigen with the E2 protein in the mixture of capture antigen. In addition, a test format using labelled HCV antigens as probe, similar to the third-generation HIV antibody screening assays, might detect IgM antibody to HCV and reduce the window period.
HEPATITIS C VIRUS
THE CASE FOR HEPATITIS C VIRUS RNA SCREENING As discussed above, nucleic acid amplification technologies (NAT) for the detection of HCV RNA are available commercially: PCR (Roche Amplicor), NASBA (nucleic acid sequence-based amplification) produced by Organon Teknika and others. All have a sensitivity below 1,000 genome equivalent/mL, which would be adequate for the detection of HCV infected individuals. However, molecular biological techniques are not routine in many diagnostic laboratories and presently are not incorporated into most blood centers, except in a limited number of associated research laboratories. Genomic detection includes three critical steps: (1) sample preparation (nucleic acid extraction) and reverse transcription from RNA to cDNA; (2) amplification using a single pair of primers or two pairs of primers in the case of "nested" amplification, and (3) detection of amplicon. To avoid carry over contamination, reagent preparation and the first two steps must be performed in separate facilities. While manufacturers have put considerable effort on the amplification steps, little has been done for sample preparation. Importantly, HCV RNA detection assays cost more than $50/test which makes it prohibitive for large-scale screening, particularly in healthy blood donors. During the past few years, evidence has been provided that several lots of anti-D Ig infected pregnant women with HCV in the late 1970s l~176 and that intravenous Ig prepared without a virus inactivation step could transmit HCV to recipients. 1H-H3 HCV RNA was detected in implicated lots. 114 These observations prompted some agencies controlling plasma derivatives to take steps towards HCV RNA screening in either the final products or plasma pools and the destruction of positive lots or pools. To avoid losses of thousands of liters of source plasma, manufacturers devised methods to detect HCV RNA in smaller pools. Various preliminary technical solutions have been proposed to circumvent the many problems raised by pool testing. First, a decrease in sensitivity proportional to the number of individual plasmas in the pool (range 100 to 1000). Second, the possibility that endogenous NAT inhibitors cause falsenegative results, although the dilution factor may limit this problem. Third, the necessity not only to devise special equipment to prepare pools containing a precise volume of each constituting sample
51
but also to introduce software able to identify individual samples included in each pool or fractions of pool. Information provided by companies in the United States and Germany, in collaboration with blood banking organizations, indicate that sensitivity and inhibitors issues could be addressed by an ultracentrifugation step prior to nucleic acid preparation. Equipment and adequate software for pool preparation and donor identification are available. However, recently, in a German Red Cross blood center less than 10% of plasma pools from 400 donations were found to be positive; the majority of positive pools were found not to contain a confirmed positive individual plasma, suggesting low specificity of NAT (Schottstedt, personal communication, 1997). Although 90% of the blood supply could be released for clinical use within 2 days, identification of positive individuals took 5 to 7 days. Irrespective of the specific technical issues involved, plasma pool screening is feasible and may be adequate for the purposes of plasma industry and associated regulatory agencies. Application of such programs to recovered plasma, which provides a substantial part of the source plasma for fractionation, will create problems for the management of blood components unless screening is anonymous. Ninety percent of the blood or blood components will be quarantined for 48 hours, decreasing by 20% the shelf-life of platelet concentrates. The remaining 10%, initially found to be positive, will be retained for 5 to 7 days by which time all platelet concentrates will be outdated. Alternatively, these products may be distributed knowing that some recipients of platelet concentrates and a smaller number of red blood cell recipients will be transfused with products later found infectious. Past experience has shown that such a situation is not acceptable ethically and legally. Consequently, HCV RNA screening of individual blood donors may fulfil the requirements of both blood component and plasma derivative providers. The assay sensitivity must be optimal, the impact of inhibitors minimal, and test results must be available within a time frame compatible with good transfusion medicine practice. Such plans cannot be implemented, therefore, until several technical problems are solved. Current commercial assays take more than 24 hours to perform in small series. Blood component output requires results in
52
JEAN-PIERRE ALLAIN
less than 8 hours with a flowthrough in the range of 500/d. Routine testing in the blood bank environment requires robust, self-contained, highly automated assays that urgently need to be developed for NAT. lt5-117Finally, the cost of HCV RNA screening is well above what the wealthiest countries can afford. The development of assays designed to simultaneously detect genomes of multiple infectious agents will help to decrease the cost. It can be argued that genomic screening is unnecessary when viral inactivation procedures are applied to blood components such as solvent-detergent to freshfrozen plasma, ultraviolet/psoralen to platelet concentrates, or various photoactivable chemicals to red cell concentrates. However, as repeatedly pointed out for plasma derivatives, decreasing the
viral burden before virucidal procedures is critical to their efficacy. Genomic screening and virucidal procedures will be complementary. CONCLUSION
The performance of HCV antibody screening has been spectacularly effective in reducing posttransfusion infection. The residual risk of HCV infection estimated to 1:20,000 to 1:200,000 will be virtually eliminated with the implementation of NAT for HCV. The possibility of multiplex NAT screening for HCV, HIV and other viral genomes adapted to the needs of blood donor screening may be a major step towards zero risk of posttransfusion infection.
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virus among blood donors: Follow-up study. Transfusion 34:527530, 1994 29. Feucht H-H, Ztllner B, Polywka S, et al: Study on reliability of commercially available hepatitis C virus antibody tests. J Clin Microbiol 33:620-624, 1995 30. Le6n P, L6pez JA, Domingo C, et al: Evaluation of laboratory assays for screening antibody to hepatitis C virus. Transfusion 33:268-270, 1993 31. Barbara JAJ, Contreras M: Non-A, non-B hepatitis and the anti-HCV assay. Vox Sang 60:1-7, 1991 32. Dow BC, Follett EAC, Jordan T, et al: Testing of blood donations for hepatitis C virus. Lancet 343:477-478, 1994 33. Vernelen K, Claeys H, Verhaert H, et al: Significance of NS3 and NS5 antigens in screening for HCV antibody. Lancet 343:853, 1994 (letter) 34. Courouce A-M, Janot C, Hepatitis Study Group: Recombinant immunoblot assay first and second generations on 732 blood donors reactive for antibodies to hepatitis C virus by ELISA. Vox Sang 61:177-180, 1991 35. Vrielink H, van der Poel CL, Reesink HW, et al: Transmission of hepatitis C virus by anti-HCV-negative blood transfusion. Vox Sang 68:55-56, 1995 36. Vrielink H, Zaaijer HL, Reesink HW, et al: Comparison of two anti-hepatitis C virus enzyme-linked immunosorbent assays. Transfusion 35:601-604, 1995 37. Uyttendaele S, Claeys H, Mertens W, et al: Evaluation of third-generation screening and confirmatory assays for HCV antibodies. Vox Sang 66:122-129, 1994 38. Bresters D, Reesink HW, Cuypers HTM, et al: Sensitivity of an anti-HCV core peptide ELISA. J Med Virol 37:187-191, 1992 39. Gabriel FG, Teo CG: Peptide based enzyme immunoassays for detecting hepatitis C antibodies in sera of people at risk. J Clin Patho147:357-359, 1994 40. Yuki N, Hayashi N, Kasahara A, et al: Hepatitis C virus replication and antibody responses toward specific hepatitis C virus proteins. J Hepatol 19:1360-1365, 1994 41. Yuki N, Hayaski N, Hagiwara H, et al: Hepatitis C virus antibodies and virus replication in asymptomatic blood donors. Vox Sang 67:280-285, 1994 42. Lelie PN, Cuypers HTM, Reesink HW, et al: Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays. J Med Virol 37:203-209, 1992 43. Bhattacherjee V, Prescott LE, Pike I, et al: Use of NS-4 peptides to identify type-specific antibody to hepatitis C virus genotypes 1, 2, 3, 4, 5, and 6. J Gen Viro176:1737-1748, 1995 44. Lesnievski RR, Boardway KM, Casey JM, et al: Hypervariable 5'-terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants. J Med Viro140:150-156, 1993 45. Lesniewski R, Okasinski G, Carrick R, et al: Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: A new marker of HCV infection closely associated with viremia. J Med Viro145:415-422, 1995 46. Le6n P, Ltpez JA, Elola C, et al: Detection of antibody to hepatitis C virus E2 recombinant antigen among samples indeterminate for anti-HCV after wide serological testing and correlation with viremia. Vox Sang 70:213-216, 1996 47. Chien DY, Choo Q-L, Ralston R, et al: Persistence of HCV despite antibodies to both putative envelope glycoproteins. Lancet 342:933, 1993 (letter)
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48. Zibert A, Schreier E, Roggendorf: Antibodies to human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment. Virology 208:653-661, 1995 49. Scarselli E, Cerino A, Esposito G, et al: Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp 70) hypervariable region 1 in viremic hepatitis C virus-infected patients. J Viro160:4407-4412, 1995 50. Jackson P, Petrik J, Alexander GJM, et al: Reactivity of synthetic peptides representing selected sections of hepatitis C virus core and envelope proteins with a panel of hepatitis C virus-positive human plasma. J Med Virol 51:67-79, 1997 51. Lau GKK, Lesniewski R, Johnson RG, et al: Immunoglobulin M and A antibodies to hepatitis C core antigen in chronic hepatitis C virus infection. J Med Viro144:1-4, 1994 52. Nagayama R, Miyake K, Tsuda F, et al: IgM antibody to a hepatitis C virus core peptide (CP14) for monitoring activity of liver disease in patients with acute or chronic hepatitis C. J Med Viro142:311-317, 1994 53. Kapprell HP, Majnak A, Medgyesi GA, et al: HCV core IgM and IgG antibodies in blood donors. Vox Sang 68:57-58, 1995 54. Busch MP, Tobler L, Quan S, 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 33:84-88, 1993 55. Evans CS, Tobler A, Polito A, et al: Comparative evaluation of supplemental hepatitis C virus antibody test systems. Transfusion 32:408-414, 1992 56. Van der Poel CL, Cuypers HTM, Reesink HW, et al: Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 337:317-319, 1991 57. Van der Poel CL, Reesink HW, Mauser-Bunschoten EP, et al: Prevalence of anti-HCV antibodies confirmed by recombinant immunoblot in different population subsets in The Netherlands. Vox Sang 61:30-36, 1991 58. Bresters D, Zaaijer HL, Cuypers HTM, et al: Recombinant immunoblot assay reaction patterns and hepatitis C virus RNA in blood donors and non-A, non-B hepatitis patients. Transfusion 33:634-638, 1993 59. Allain J-P, Rankin A, Kuhns MC, et al: Clinical importance of HCV confirmatory testing in blood donors. Lancet 339:1171-1172, 1992 60. Tobler LH, Busch MP, Wilber J, et al: Evaluation of indeterminate c22-3 reactivity in volunteer blood donors. Transfusion 34:130-134, 1994 61. Follett EAC, Dow BC, McOmish F, et al: HCV confirmatory testing of blood donors. Lancet 339:372, 1992 62. Boudart D, Lucas JC, Adjou C, et al: HCV confirmatory testing of blood donors. Lancet 339:372, 1992 63. Chan SW, Simmonds P, McOmish F, et al: Serological responses to infection with three different types of hepatitis C virus. Lanct 338:1391, 1991 (letter) 64. 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 30:931-934, 1992 65. Le6n P, L6pez JA, Domingo CJ, et al: Serological studies and hepatitis C virus RNA in serum samples from blood donors with indeterminate results in second-generation recombinant immunoblot assay. Vox Sang 66:245-246, 1994 66. Dussaix E, Charnaux N, Laurent-Puig P, et al: Analysis of sera indeterminate by Ortho-HCV RIBA-2 by using three
54
confirmatory assays for anti-hepatitis C virus antibody. J Clin Microbiol 32:2071-2075, 1994 67. Garc~a-Samaniego J, Soriano V, Silva E, et al: Significance of HCV RIBA-2 indeterminate results in high-risk individuals: assessment by a new third-generation RIBA assay and PCR. Vox Sang 66:148-149, 1994 68. Pawlotsky J-M, Fleury A, Choukroun V, et al: Significance of highly positive c22-3 "indeterminate" second generation hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) and resolution by third-generation HCV RIBA. J Clin Microbio121:1357-1359, 1994 69. Mutimer DJ, Harrison RF, O'Donnell KB, et al: Hepatitis C in asymptomatic British blood donors with indeterminate seropositivity. Br Med J 309:847-848, 1994 70. Yuki N, Hayaski N, Hagiwara H, et al: Hepatitis C virus antibodies and virus replication in asymptomatic blood donors. Vox Sang 67:280-285, 1994 71. Rossini A, Gazzola GB, Ravaggi A, et al: Long-term follow-up of an infectivity in blood donors with hepatitis C antibodies and persistently normal alanine amino-transferase levels. Transfusion 35:108-111, 1995 72. Zaaijer HL, Vrielink PJ, van Excel-Oehlers HTM, et al: Confirmation of hepatitis C infection: A comparison of five immunoblot assays. Transfusion 34:603-607, 1994 73. Damen M, Zaaijer HL, Cuypers H, et al: Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus. Transfusion 35:745-749, 1995 74. Buffet C, Charnaux N, Lanrent-Puig, et al: Enhanced detection of antibodies to hepatitis C virus by use of a third-generation recombinant immunoblot assay. J Med Virol 43:259-261, 1994 75. Dow BC, Follett EAC, Munro H, et al: Failure of 2ndand 3rd-generation HCV ELISA and RIBA to detect HCV polymerase chain reaction-positive donations. Vox Sang 67:236237, 1994 76. Pawlotsky J-M, Bastie A, Pellet C, et al: Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay. J Clin Microbio134:80-83, 1996 77. Damen M, Vrielink H, Lelie PN, et al: Incorrect diagnosis of hepatitis C with RIBA-3. Vox Sang 69:358, 1995 (letter) 78. Allain J-P: Can recombinant immunoblot assay generate an erroneous diagnosis of hepatitis C virus infection? Transfusion 36:476-477, 1996 79. Atrah HI, Hutchinson F, Gough D, et al: Hepatitis C virus seroconversion rate in established blood donors. J Med Virol 46:329-333, 1995 80. Atrah HI, Ala FA, Ahmed MM, et al: Unexplained hepatitis C virus antibody seroconversion in established blood donors. Transfusion 36:339-343, 1996 81. Lee SR, Nelles MJ, Diunello R, et al: Can recombinant immunoblot assay generate an erroneous diagnosis of hepatitis C virus infection? Transfusion 36:477, 1996 (letter) 82. Das PC, Heethuis A, McShine RL, et al: Supplemental HCV antibody assay in blood donors by RIBA and line immunoassay. Transf Med 4:173-175, 1994 83. Allain J-P, Kitchen A, Aloysius S, et al: Safety and efficacy of hepatitis C virus antibody screening of blood donors with two sequential screening assays. Transfusion 36:401-405, 1996 84. Farci P, Alter HJ, Govindarajan S, et al: Lack of
JEAN-PIERRE ALLAIN
protective immunity against reinfection with hepatitis C virus. Science 258:135-140, 1992 85. Prince A, Brotman B, Huima T, et ai: Immunity in hepatitis C infection. J Inf Dis 165:438-443, 1992 86. Sugitani M, Inchausp6 G, Shindo M, et al: Sensitivity of serological assays to identify blood donors with hepatitis C viraemia. Lancet 339:1018-1019, 1992 87. Vrielink H, van der Poe1 CL, Reesink HW, et al: Look-back study of infectivity of anti-HCV ELISA-positive blood components. Lancet 345:95-96, 1995 88. Vrielink H, van der Poe1 CL, Reesink HW, et al: Transmission of hepatitis C virus by anti-HCV-negative blood transfusion. Vox Sang 68:55-56, 1995 89. Weiner AJ, Kuo G, Bradley DW, et al: Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 335:1, 1990 (letter) 90. Kaneko S, Unoura M, Kobayashi K, et al: Detection of serum hepatitis C virus RNA. Lancet 335:976, 1990 (letter) 91. Garson JA, Ring C, Tnke E et al: Enhanced detection by PCR of hepatitis C virus RNA. Lancet 336:878-879, 1990 92. Garson JA, Tedder RS, Briggs M, et al: Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet 335:1419, 1990 (letter) 93. Zanetti AR, Tanzi E, Zehender G, et al: Hepatitis C virus RNA in symptomless donors implicated in post-transfusion non-A, non-B hepatitis. Lancet 336:447, 1990 (letter) 94. Garson JA, Clewley JE Simmonds E et al: Hepatitis C viraemia in United Kingdom blood donors. Vox Sang 62:218223, 1992 95. Gretch DR, Wilson JJ, Carithers RL, et al: Detection of hepatitis C virus RNA: Comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR. J Clin Microbiol 31:289-291, 1993 96. Gretch DR, de la Corazon R, Robert MT, et al: Assessment of hepatitis C viremia using molecular amplification technologies: Correlations and clinical implications. Ann Int Med 123:321-329, 1995 97. Ulrich PP, Romeo JM, Lane PK, et al: Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferase. J Clin Invest 86:1609-1614, 1990 98. Prati D, Capelli C, Bosoni P, et al: Determination of hepatitis C virus RNA in the serum by the Amplicor HCV PCR kit. Vox Sang 67:112-114, 1994 99. Bresters D, Cuypers HTM, Reesink HW, et al: Comparison of quantitative cDNA-PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophilia patients participating in a controlled interferon trial. J Med Viro143:262-268, 1993 100. Manzin A, Bagnarelli P, Menzo S, et al: Quantitation of hepatitis C virus genome molecules in plasma samples. J Clin Microbio132:1939-1944, 1994 101. McGuinness PH, Bishop GA, Lien A, et al: Detection of serum hepatitis C virus RNA in HCV antibody-seropositive volunteer blood donors. Hepatology 18:485-490, 1993 102. Nalpas B, Romeo R, Pol S, et al: Serum hepatitis C virus (HCV) RNA: A reliable tool for evaluating HCV-related liver disease in anti-HCV positive blood donors with persistently
HEPATITIS C VIRUS
normal alanine aminotransferase values. Transfusion 35:750753, 1995 103. French study group for the standardization of hepatitis C virus PCR: Improvement of hepatitis C virus RNA polymerase chain reaction through a multicentre quality control study. J Virol Meth 49:79-88, 1994 104. Damen M, Cuypers HTM, Zaaijer HL, et al: Internal collaborative study on the second EUROHEP HCV RNA reference panel. J Virol Meth 58:175 - 185, 1996 105. Japanese Red Cross non-A, non-B Hepatitis Research Group: Effect of screening for hepatitis C virus antibody and hepatitis B virus core antibody on incidence of post-transfusion hepatitis. Lancet 338:1040-1041, 1991 106. Donahue JG, Munoz A, Ness PM, et al: The declining risk of post-transfusion hepatitis C virus infection. N Engl J Med 327:369-373, 1992 107. Gonzales A, Esteban JI, Madoz P, et al: Efficacy of screening donors for antibodies to the hepatitis C virus to prevent transfusion-associated hepatitis: Final report of a prospective trial. Hepatology 22:439-445, 1995 108. Schreber GB, Busch MP, Kleinman SH, et al: The risk of transfusion-transmitted viral infections. N Eng J Med 334: 1685-1690, 1996 109. Power JP, Lawlor E, Davidson F, et al: Hepatitis C viraemia in recipients of Irish intravenous anti-D immunoglobulin. Lancet 345:1211-1213, 1995
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110. Dittmann S, Roggendorf M, Durkop J, et al: Long-term persistence of hepatitis C virus antibodies in a single source outbreak. J Hepatol 13:323-324, 1991 111. Pawlotsky JM, Bouvier M, Deforges L, et al: Chronic hepatitis C after high dose intravenous immunoglobulin. Transfusion 34:86-87, 1994 112. Yu MW, Mason BL, Guo ZP, et al: Hepatitis C transmission associated with intravenous immunoglobulins. Lancet 345:1173-1174, 1994 113. Nubling CM, Willkommen H, Lower J: Hepatitis C transmission associated with intravenous immunoglobulins. Lancet 345:1174, 1995 (letter) 114. Yu MYW, Mason BL, Tankersley DL: Detection and characterization of hepatitis C virus RNA in immune globulins. Transfusion 34:596-602, 1994 115. Shindo M, Di Bisceglie AM, Silver J, et al: Detection and quantitation of hepatitis C virus RNA in serum using the polymerase chain reaction and a colorimetric enzymatic detection system. J Virol Methods 48:65-72, 1994 116. Van Doom LJ, Kleter B, Voermans J, et al: Rapid detection of hepatitis C virus RNA by direct capture from blood. J Med Viro142:22-28, 1994 117. Petrik J, Pearson GJM, Allain JP: High throughput PCR detection of HCV based on semiantomated multisample RNA capture. J Virol Meth 64:147-159, 1997
EPATITIS C VIRUS (HCV), the main etiologic agent of the clinical entity formerly known as non-A, non-B hepatitis, was discovered in 1989 by a group of collaborating scientists from Chiron and the Centers for Disease Control. 1 Recombinant proteins were produced and used for the detection of antibodies to the new agent. 2 This assay has proven to be extremely valuable for the diagnosis of HCV infection. Because non-A, non-B hepatitis was by far the most common transfusion transmitted infection, the newly developed test was applied to the screening of blood donors. Subsequently, significant improvements were made in test performance to achieve the demanding requirements of blood donor screening. During the past 7 years, a continuous effort has been made by Chiron, its licensees, and research units to improve test performance both in sensitivity and specificity, as had occurred previously with antibody screening assays for human immunodeficiency virus (HIV) or human T cell lymphotropic virus H I . However, access to the HCV patent has been restricted to a limited number of diagnostic companies. The usual competition for quality and price from which the public benefits did not take place to the full extent that would be desirable. Very early on, it was recognized that antibody to HCV was more difficult to detect and its clinical significance more difficult to interpret than other viral markers such as antibodies to HIV. In the absence of an HCV antigen assay, the recent availability of genomic amplification techniques has provided the tools to complement antibody screening. This review summarizes the progress made during the relatively short history of HCV antibody screening and confirmation tests as well as the critical role played by genomic amplification in the interpretation of screening test results. The extent of these difficulties might lead, in a not too distant future, to a genomic screening test for
H
From the Division of Transfusion Medicine, Department of Haematology, University of Cambridge, Cambridge, UK. Address reprint requests to Professor Jean-Pierre Allain, MD, PhD, Division of Transfusion Medicine, East Anglia Blood Centre, Long Road, Cambridge, CB2 2PT, United Kingdom. Copyright 9 1998 by W.B. Saunders Company 0887-7963/98/1201-000553.00/0
46
HCV to virtually eliminate the risk of HCV transmission by blood product transfusion. THE HISTORY OF HEPATITIS C VIRUS ANTIBODY SCREENING
First Generation Screening Tests The isolation by immunoscreening of a small fragment of the HCV nonstructural protein 4 (NS4) led to the development of a prototype radioimmunoassay (RIA) based on the recombinant 5-1-1 protein. 2 The high proportion of clinical and posttransfusion non-A, non-B hepatitis cases reacting with protein 5-1-1 provided strong evidence that these antibodies were a specific marker of this elusive hepatitis viral agent. Subsequently, a solid body of data became available clearly showing the presence of antibodies to 5-1-1 in many cases of non-A, non-B hepatitis. 3,4 A larger and more effective recombinant antigen called cl00 was then used in an RIA that showed that antibodies to the putative viral agent were present both in donors and in recipients implicated in post-transfusion non-A, non-B hepatitis. In addition, an American population of blood donor showed a rate of positivity ranging from 0.5% to 8%. 5 Antibody to 5-1-1 was correlated with both alanine amino transferase levels and the presence of anti-HBc that had previously been associated with non-A, non-B post-transfusion hepatitis. Very quickly, insufficiencies in the performance of the cl00-based enzyme-linked immunosorbent assay (EIA) ~ was pointed out by many investigators. 6-8 Sensitivity was insufficient as a large proportion of clinically defined non-A, non-B hepatitis were anti-cl00 negative, and antibody was often detectable only weeks after the clinical development of hepatitis. 3 Specificity was impaired by a variety of factors. 9q2 The awaited development of assays using the recombinant 5-1-1 and cl00 antigens coated on a strip of nitrocellulose to capture antibodies detected in an enzyme immunoblot format provided the first means of confirmation. 13,14 Other manufacturers used confirmatory blocking or synthetic peptide assays. 15 Although the vast majority of clinically selected individuals with non-A, non-B hepatitis, or those at high risk of blood borne viral infection such as hemophiliacs or Transfusion Medicine Reviews, Vo112, No 1 (January), 1998: pp 46-55
HEPATITIS C VIRUS
47
tially higher rate of seropositivity was found in high-risk groups, such as hemophiliacs. 24-26 In blood donors who were positive with the firstgeneration assay, 40% to 60% repeatedly reactive samples did not react with the second-generation assays; these negative samples could not be confirmed. 27-29 In untested blood donors, secondgeneration screening assays based on recombinant proteins showed higher sensitivity and specificity.3~ In the United Kingdom, EIA positivity rate ranged from 0.5% to 1% and from 0.2% to 0.4% with firstand second-generation tests, respectively.31 However, the specificity of second-generation screening assays was still far from optimal because nearly 50% of reactive samples could not be confirmed. To overcome this difficulty, several manufacturers attempted to use screening assays that included synthetic peptides from highly immunogenic portions of the core, NS3, NS4, and NS5 regions. They considerably improved specificity but lacked sensitivity.3~ These features were not surprising as synthetic peptides do not contain the potentially reactive impurities from the bacteria used to produce recombinant proteins but have a restricted spectrum of epitopes derived from short segments of a model virus, not reflecting the considerable genetic diversity of HCV.
intravenous drug abusers, were confirmed positive, l some false-positive results were obtained) 6 Nineteen percent to 33% seropositive blood donors were confirmed, indicating a high rate of false positivity. 15,17,~8The test's lack of specificity and relatively poor clinical correlation were taken as sufficient arguments to postpone the screening of blood donors in some countries.~9.2~
Second-Generation Screening Tests A second generation of anti-HCV EIA screening was developed and used in 1991. In some assays, recombinant HCV proteins, particularly from the putative structural antigens c-22 core and nonstructural c-33 from the NS3 region, were added to the NS4 cl00 used in the first-generation assays. In other assays, synthetic peptides derived from the immunogenic sequences of the core, NS3 and NS4 from the prototype virus were used. 2~ As shown in Table 1, five tests became available, two of them totally, or partially, based on synthetic peptides. The introduction of these new antigens nearly doubled the anti-HCV positive rate and reduced the window period by several weeks in patients with acute non-A, non-B hepatitis, explaining many of the antibody/clinical discrepancies observed with the first-generation screening assays. 22,23A substan-
Table 1. HCV Antibody Screening and Confirmatory Assays Recombinant Antigens Core
NS3
Assay Ortho 1st generation EIA Abbott 1st generation EIA Ortho 2d generation EIA Abbott 2d generation EIA Murex EIA UBI-EIA SANOFI EIA Ortho 3d generation EIA Abbott 3d generation EIA RIBA.1 RIBA.2 RIBA.3 INNO-LIAt Murex WB Dediscan (Sanofi)
NS4 c100
+ + +
Synthetic Peptides
NS5 5-1-1
Core
E2/NS1
+ + + +
+ + +
+ c200" c200
+
+ +
+
+ +
+ +
NS5
+ +
+
+ + +
+ +
NS4
+ + • 4
+
+ +
+ +
+
Note. c100, 5-1-1 are designations of recombinant proteins initially used by Chiron Corporation. *c200 is a fusion protein of the NS3 and NS4 recombinant proteins. tlNNO-LIA from Innogenetics uses four separate core peptides of HCV. Other Abbreviations Used: NS3, nonstructural protein 3; NS4, nonstructural protein 4; NS5, nonstructural protein 5; E2, envelope 2; WB, Western blot; RIBA, recombinant immuno-blot assay; EIA, enzyme-linked immunosorbant assays; UBI-EIA, United Biochemical Incorporation.
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Third-Generation Screening Tests In 1993, a third generation of anti-HCV screening assays was introduced (Table 1). The main feature was the addition of a recombinant portion of the NS5 region of the HCV polyprotein. In some assays, the NS3 and the NS4 antigens were presented as a fusion protein c200. These changes did not significantly improve the sensitivity of antiHCV detection, but most of the added sensitivity was related to an improvement in anti-NS3 detection rather than a n t i - N S 5 . 32-34 In contrast, the addition of the NS5 antigen reduced the specificity obtained with the second-generation EIA. 35,36Most additional reactive samples that reacted only with the NS5 protein on the immunoblot confirmatory assay did not contain HCV RNA, suggesting falsepositive reactions. 36,37 For these reasons, secondgeneration assays were considered to be of superior performance and prefered to third-generation tests by many investigators.
Other Immunoglobulin G Detection Assays Commercial HCV antibody screening assays are all based on recombinant proteins from core, NS3, NS4, or NS5 proteins of various length, produced in various expression systems. Some also use core peptides coated on microtitre plates or polystyrene beads. The test format, as in screening assays for other viral markers, is uniformly an indirect antiglobulin EIA. As early as 1991, the high immunogenicity and high level of sequence conservation of the N-terminus of the core protein of HCV led to the development of core only or core plus other synthetic peptide-based EIAs. 21,38;39 Data collected indicated that HCV was relatively poorly immunogenic, antibody titres being often low and to some extent parallel to the level of viremia. 4~ In infected individuals, HCV elicited an immunoglobulin G (IgG) immune response to the various antigens over variable and often prolonged periods. In some individuals, antibodies, often to NS4, but also on occasion to core or NS3, did not occur or reach detectable levels. 42 In addition, HCV has a high level of genomic and antigenic variability that may be reflected in antibody detection to be used for serologic genotyping. 43 These peculiar features of HCV infection explain the relative failure of screening assays based on short peptide sequences covering a limited number of epitopes derived from a single (not always the most prevalent) viral sequence.
JEAN-PIERRE ALLAIN
Unlike the HIV antibody assays, the HCV envelope proteins are remarkably absent from the screening panel of antigens. The E2 glycoprotein has been the focus of considerable interest both because of its high level of antigenicity and its high variability in its N-terminus portion where hypervariable regions 1 and 2 are located (HVR1 and 2). E2 has been expressed in a fully glycosylated form in mammalian cells 44 and used in antibody assays. 45,46 Similar to other HCV recombinant antigens, a high (->90% in HCV RNA-positive patients) frequency of reactivity was observed. The low level of expression and higher cost of this antigen probably explain its absence from the screening assays. Sequence analysis and studies of specific E2 peptide reactivity indicated a small number of cross-reactive epitopes outside of the HVR regions and a largely restricted reactivity of the HVR highly immunogenic epitopes. 47-5~ The discrepancy observed between the high prevalence of anti-recombinant E2 and the low prevalence of anti-E2 peptides suggest the importance of conformational and/or carbohydrate-related epitopes.
lgM and IgA Anti-Hepatitis C Virus Estimations IgM anti-core peptides of HCV have been tested for in patients with HCV infection and donor screening with 23% to 86% of patients with chronic HCV-related liver disease found to be positive for IgM anti-HCV, 51,52and 33% in asymptomatic HCV carriers. 4~ Screening blood donors for IgM anti-HCV yielded a prevalence three times lower than that for IgG anti-HCV; although no confirmation assay for IgM is available. IgA antibody to HCV core peptide could not be detected in one study of patients with chronic liver disease. 51 CONFIRMATION OF HEPATITIS C VIRUS ANTIBODY SCREENING
As observed with other antibody assays, the positive predictive value (PPV) for the diagnosis of past or present infection decreased sharply with the prevalence of true infection in the studied population. PPV is particularly low in populations of healthy people at low risk of infection such as blood donors. Supplementary tests are necessary to differentiate between true anti-HCV antibodies and false positivity. To be effective, confirmatory assays should differ from the screening assay not only in the test format but also the antigens used for
HEPATITIS C VIRUS
detection. In the case of HCV antibody, these basic rules have been only partially followed, creating some difficulty in interpretation of test results. The first anti-HCV confirmatory assay and its subsequent modifications were called recombinant immunoblot assays (RIBA). Antigens used in the first generation (RIBA.1) were 5-1-1- and cl00coated on a strip of nitrocellulose together with an IgG procedural control and SOD as the main source of false positivity. Reactivity to both antigens was required for positivity. Similarly to the firstgeneration screening assays, the sensitivity and specificity of RIBA.1 were low and the disadvantages of using the same antigens as the screening assay in the same indirect EIA format were noted. 54-57 cl00 expressed in Eschericheria coli instead of yeast and synthetic peptides derived from the cl00 sequence were used by manufacturers to improve specificity.55,s8 Together with the second-generation screening assays, RIBA.1 was soon replaced by RIBA.2. Three screening antigens (c22, c33, and cl00) and 5-1-1 were coated on the nitrocellulose strip. Confirmation was defined as the reactivity of at least two of these four HCV antigens, although many investigators considered 5-1-1 and cl00 reactivity as insufficient to define confirmation because essentially they were overlapping epitopes. 54.55 Nonetheless, sensitivity and diagnostic value improved considerably.23,56 When applied to a population of blood donors positive with firstgeneration screening assay, RIBA.2 increased the confirmation rate slightly and many indeterminate results became negative. 55,57In patients chronically infected by HCV (HCV RNA-positive), RIBA.2 reacted with three antigens in 60%, two antigens (c22 and c33) in 25% to 30%, and with only one antigen (either c22 or c33) in 6% to 10% of samples .58 In second-generation screening test positive blood donors, 50% were RIBA.2 negative, 25% confirmed, and 25% indeterminate. 59,6~ Fifty percent to 70% of indeterminate samples were reacting with the c22 (core) antigen, the rest nearly equally distributed between c33 and cl00 reactivity.59,6~,62The significance of indeterminate RIBA.2 results were variably interpreted: false positivity, residual antibody postrecovery of HCV infection, partial antibody pattern related to HCV subtypes, 63 or true persistent infection confirmed by the presence of HCV RNA. The general opinion was that most were false-positive because they
49
were either not reacting with other screening ass a y s , 59,60,64,65 o r with other confirmatory assays. 15'60'66-68 In addition, they were often nonreproducible over time, 62 were negative for HCV RNA, 59-61 and were not associated with clinical or histologic evidence of liver disease. 69 However, 10% to 25% of c22 indeterminate samples contained HCV RNA, suggesting that some individuals generate detectable antibodies only to this antigen, either because of a low level of viral replication7~ or because of a different HCV subtype. 63 This situation is more frequent in patients receiving immunosuppressive treatment. 68 HCV RNA has seldom been found in anti-c33 and exceptionally in anti-cl00 indeterminate samples.S4,58,65,71 The relative lack of specificity of RIBA.2 was considerably reduced with RIBA.3, which detected antibody to HCV core by specific peptides. 37,72-74 Sensitivity was also improved by a better detection of anti-NS3 antibodies. 73,74 Many HCV RNAnegative samples reacted exclusively with the NS5 antigen revealing that false-positive screening tests were often related to this antigen. As with cl00, virtually no anti-NS5 indeterminate samples were found concomitantly HCV RNA-positive. 75 Although RIBA.3 yielded approximately 10% indeterminate results, 76 some false-positive results were still obtained, as they were not detected by alternative confirmatory assays except when HCV RNApositive. 72 Indeterminate results are not the only difficulty in the interpretation of RIBA or other confirmatory assays. A few cases of blood donors reacting with two antigens such as c33 or NS5 or NS4 but with no other epidemiological or clinical evidence of HCV infection have been described. 77-8~Analysis of such data suggest that such confirmed antibody results are, in fact, false-positive. Reasons for these observations are unclear. 81 These indeterminate test results clearly indicate that, contrary to that found with HIV serology, HCV screening and confirmatory data are in some cases insufficient to provide reliable information for a correct diagnosis and appropriate patient counselling. The simultaneous development of HCV RNA detection methods by genomic amplification provides critical additional information. The classical confirmatory scheme, which includes initial screening assay, repeat screening assay in duplicate, confirmatory assay of repeatedly
50
JEAN-PIERRE AL/AIN
reactive samples, has been challenged on the basis of higher efficacy and improved cost-effectiveness by the following algorithm: initial screening EIA-1, repeat screening EIA-1, repeat screening EIA-2, provided the second screening assay uses different antigens than the first o n e . 39,82,83
HEPATITIS C VIRUS RNA DETECTION AND CONFIRMATION
HCV is a persistent virus in most infected individuals. 3 Approximately 10% of individuals seem to recover from the infection as HCV is not detected in serum or liver and both humoral and cellular immunity is present in the host. However, protection is fragile and superinfecfion with another viral strain or possibly the same viral strain can occur naturally or experimentally. 84,85 During the window period, circulating viral genome is the only detectable marker. There is also evidence that HCV can chronically circulate without detectable antibody. HCV transmission by transfusion of antibody positive units is correlated with the presence of HCV RNA detected by sensitive genomic amplification methods, s6-8s HCV RNA detection has a critical role to play in the diagnosis of HCV infection and the confirmation process of positive screening results, particularly in a low prevalence population. HCV genomic detection can be done by direct hybridization (Chiron branched DNA, bDNA) or nucleic acid amplification using the polymerase chain reaction (PCR). bDNA is quantitative but relatively insensitive (1 • 105 genome equivalents/ mL) and inadequate for the purpose of confirmation. Amplification methods such as PCR are applied to cDNA after nucleic acid extraction and reverse transcription. It can be performed with either single amplification step or in a nested format which requires two primer pairs and increases the risk of contamination. 8996A sensitivity below 1,000 genome equivalent/mL can be achieved, which seems sufficient for clinical purposes, and kits are commercially available. 97-99In high- and low-risk groups, symptomatic or asymptomafic, with normal or increased transaminases level, 80% to 95% of confirmed HCV antibodypositive individuals are HCV carriers. Viremia ranges from 1 • 102 to 1 • 107/mL of blood with large fluctuations seen over time in the same patient, but most patients are consistently vire-
mic. 25,96,100 This very high prevalence of HCV
viraemia is useful in the confirmation process. From a clinical point of view, HCV RNA is the highest single predictive marker of HCV infection. HCV RNA positivity has been shown to be highly predictive of infectivity by transfusion. 101,102 For confirmatory purposes, HCV genomic amplification methods, although commercially available, present technical difficulties and are associated with the risk of false-positive results, unless performed in a suitable environment including the physical separation of reagent, sample preparation, and amplification steps. False-negative results can also be obtained when endogenous inhibitors are present, when anticoagulants like heparin are used to collect the samples. It is recommended that tests be performed in reference laboratories using experienced personnel. 1~176 Genomic amplification is necessary to the confirmation process in a limited number of situations, such as an indeterminate confirmatory result or positivity with only two reactive antigens. However, for the purpose of counselling, the absence of viremia suggests strongly a low or no risk of transmission and high probability of the individual being amongst the 10% who recover from HCV infection. CLINICAL RELEVANCE OF ANTI-HEPATITIS C VIRUS SCREENING
Screening for HCV antibody has provided the means to diagnose approximately 85% of non-A, non-B hepatitis during the postacute and chronic phases. The third-generation assays in current use do not detect HCV during the window period that can last for several weeks, and rare cases of antibody-negative chronic HCV carders. Donors in these situations constitute the source of residual post-transfusion infections. Blood donor anti-HCV screening has reduced dramatically the incidence of post-transfusion non-A, non-B hepatitis, l~176 Antibody detection might still be improved with the use of an assay with higher sensitivity to include individuals with low viremia who produce very low levels of antibody: This objective could be reached by replacing the NS5 antigen with the E2 protein in the mixture of capture antigen. In addition, a test format using labelled HCV antigens as probe, similar to the third-generation HIV antibody screening assays, might detect IgM antibody to HCV and reduce the window period.
HEPATITIS C VIRUS
THE CASE FOR HEPATITIS C VIRUS RNA SCREENING As discussed above, nucleic acid amplification technologies (NAT) for the detection of HCV RNA are available commercially: PCR (Roche Amplicor), NASBA (nucleic acid sequence-based amplification) produced by Organon Teknika and others. All have a sensitivity below 1,000 genome equivalent/mL, which would be adequate for the detection of HCV infected individuals. However, molecular biological techniques are not routine in many diagnostic laboratories and presently are not incorporated into most blood centers, except in a limited number of associated research laboratories. Genomic detection includes three critical steps: (1) sample preparation (nucleic acid extraction) and reverse transcription from RNA to cDNA; (2) amplification using a single pair of primers or two pairs of primers in the case of "nested" amplification, and (3) detection of amplicon. To avoid carry over contamination, reagent preparation and the first two steps must be performed in separate facilities. While manufacturers have put considerable effort on the amplification steps, little has been done for sample preparation. Importantly, HCV RNA detection assays cost more than $50/test which makes it prohibitive for large-scale screening, particularly in healthy blood donors. During the past few years, evidence has been provided that several lots of anti-D Ig infected pregnant women with HCV in the late 1970s l~176 and that intravenous Ig prepared without a virus inactivation step could transmit HCV to recipients. 1H-H3 HCV RNA was detected in implicated lots. 114 These observations prompted some agencies controlling plasma derivatives to take steps towards HCV RNA screening in either the final products or plasma pools and the destruction of positive lots or pools. To avoid losses of thousands of liters of source plasma, manufacturers devised methods to detect HCV RNA in smaller pools. Various preliminary technical solutions have been proposed to circumvent the many problems raised by pool testing. First, a decrease in sensitivity proportional to the number of individual plasmas in the pool (range 100 to 1000). Second, the possibility that endogenous NAT inhibitors cause falsenegative results, although the dilution factor may limit this problem. Third, the necessity not only to devise special equipment to prepare pools containing a precise volume of each constituting sample
51
but also to introduce software able to identify individual samples included in each pool or fractions of pool. Information provided by companies in the United States and Germany, in collaboration with blood banking organizations, indicate that sensitivity and inhibitors issues could be addressed by an ultracentrifugation step prior to nucleic acid preparation. Equipment and adequate software for pool preparation and donor identification are available. However, recently, in a German Red Cross blood center less than 10% of plasma pools from 400 donations were found to be positive; the majority of positive pools were found not to contain a confirmed positive individual plasma, suggesting low specificity of NAT (Schottstedt, personal communication, 1997). Although 90% of the blood supply could be released for clinical use within 2 days, identification of positive individuals took 5 to 7 days. Irrespective of the specific technical issues involved, plasma pool screening is feasible and may be adequate for the purposes of plasma industry and associated regulatory agencies. Application of such programs to recovered plasma, which provides a substantial part of the source plasma for fractionation, will create problems for the management of blood components unless screening is anonymous. Ninety percent of the blood or blood components will be quarantined for 48 hours, decreasing by 20% the shelf-life of platelet concentrates. The remaining 10%, initially found to be positive, will be retained for 5 to 7 days by which time all platelet concentrates will be outdated. Alternatively, these products may be distributed knowing that some recipients of platelet concentrates and a smaller number of red blood cell recipients will be transfused with products later found infectious. Past experience has shown that such a situation is not acceptable ethically and legally. Consequently, HCV RNA screening of individual blood donors may fulfil the requirements of both blood component and plasma derivative providers. The assay sensitivity must be optimal, the impact of inhibitors minimal, and test results must be available within a time frame compatible with good transfusion medicine practice. Such plans cannot be implemented, therefore, until several technical problems are solved. Current commercial assays take more than 24 hours to perform in small series. Blood component output requires results in
52
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less than 8 hours with a flowthrough in the range of 500/d. Routine testing in the blood bank environment requires robust, self-contained, highly automated assays that urgently need to be developed for NAT. lt5-117Finally, the cost of HCV RNA screening is well above what the wealthiest countries can afford. The development of assays designed to simultaneously detect genomes of multiple infectious agents will help to decrease the cost. It can be argued that genomic screening is unnecessary when viral inactivation procedures are applied to blood components such as solvent-detergent to freshfrozen plasma, ultraviolet/psoralen to platelet concentrates, or various photoactivable chemicals to red cell concentrates. However, as repeatedly pointed out for plasma derivatives, decreasing the
viral burden before virucidal procedures is critical to their efficacy. Genomic screening and virucidal procedures will be complementary. CONCLUSION
The performance of HCV antibody screening has been spectacularly effective in reducing posttransfusion infection. The residual risk of HCV infection estimated to 1:20,000 to 1:200,000 will be virtually eliminated with the implementation of NAT for HCV. The possibility of multiplex NAT screening for HCV, HIV and other viral genomes adapted to the needs of blood donor screening may be a major step towards zero risk of posttransfusion infection.
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