Review. Laboratory Methods for Diagnosis and Management of Hepatitis C Virus Infection

Transcription

1 Laboratory Methods for Diagnosis and Management of Hepatitis C Virus Infection Muhammad Amjad, PhD, 1* Varsha Moudgal, MD, 2 Muhammad Faisal, MD 3 ABSTRACT Hepatitis C virus (HCV) infection is associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma. This review summarizes the pathogenesis and significance of serological and molecular-based assays in the diagnosis and management of HCV infection. After reading this article, readers should be able to describe laboratory tests used in the diagnosis and management of HCV infection. They Hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses, and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 1-4 Approximately 2.7 million people in the United States and 170 million people worldwide are thought to harbor persistent HCV infection. 5,6 HCV causes chronic infection due to its ability to replicate, spread, and rapidly mutate in the host. Chronic HCV infection presumably involves the emergence of quasi-species and deficient immune response to the infection; however, the basis of the inadequate immunemediated virus eradication is not known in detail. 1 DOI: /LMASROYD8BRS0GC9 Abbreviations HCV, Hepatitis C virus; ORF, open reading frame; UTRs, untranslated regions; ALT, alanine aminotransferase; EIA, enzyme immunoassay; CIA, chemiluminescence immunoassay; FDA, United States Food and Drug Administration; HIV, human immunodeficiency virus; RT-PCR, reverse transcriptase polymerase chain reaction; NAT, nucleic acid tests; TMA, transcription-mediated amplification; bdna, branchedchain DNA; SVR, sustained virologic response; E2, envelope 2; NS, nonstructural; PKRBD, protein kinase resource binding domain; ISDR, interferon sensitivity-determining region; NLS, nuclear localization signal; IL28B, human interleukin-28b 1 Department of Clinical Laboratory Sciences, Marshall University, Huntington, West Virginia, 2 Saint Joseph Mercy Hospital, Ann Arbor, Michigan, 3 Department of Pathology and Microbiology, Aga Khan University, Karachi, Pakistan *To whom correspondence should be addressed. amjad@marshall.edu should also be able to describe the general principles for selecting the most appropriate laboratory test for diagnosis, therapeutic decision making, and assessment of virologic response to therapy. Keywords: hepatitis C virus, hepatitis, hepatocellular carcinoma, molecular diagnostics HCV is a single-stranded RNA virus approximately 9.6 kb long genomic sequence, consisting of a single open reading frame (ORF) flanked by 5' and 3' untranslated regions (UTRs). The HCV ORF encodes a single polypeptide of 3037 to 3800 amino acids in length and is post-translationally modified to produce at least 10 different proteins in the following order: 5'-C-E1-E2-p7-NS2-NS3- NS4A-NS4B-NS5A-NS5B-3'. C is core, E1 and E2 are envelope proteins, and p7 is an ion channel, and NS2, NS3, NS4A, NS4B, NS5A, and NS5B are nonstructural proteins (Figure 1). 7 Six major and several minor genotypes of HCV have been identified worldwide. 8,9 Sequence homology among the different genotypes is 66%-69%. Homology between subtypes of the same genotype is 77%-80%. 10,11 Genotypes 1, 2, and 3 predominate in Japan, Western Europe, and North America. In the United States, 73.3% of patients chronically infected with HCV have genotype 1. Type 4 has been found in Central and Northern Africa and in the Middle East. Type 5 has been found in South Africa and the Middle East, and type 6 has been identified in China and Southeast Asia. 12 Along with these major genotypes, several subtypes and numerous quasi-species with minor genetic differences can emerge during the course of infection and treatment with antiviral agents. These quasi-species are defined by a sequence variability of less than 10% and continue to evolve in an individual over time, whereas the genotype does not change. Research results have suggested that the infecting genotype, quasi-species variations, and viral load correlate with the disease severity, degree of liver damage, and response to interferon therapy Lab Medicine Fall 2013 Volume 44, Number Review_Amjad.indd 292

2 Capsid Envelope Thio-Protease Ion Channel Membrane Vesicles Ser-prot, N3 cofactor NTPase/helicase IFN-α Resistance Replication RdRp F 5 C E1 E2 p7 NS2 NS3 NS4a NS4b NS5a NS5b 3 Host Signalase Physical Map of HCV Genome Laboratory Methods for Diagnosing Hepatitis C Virus Infection A reliable culture method for HCV is not currently available. Laboratory assays currently available to diagnose and manage HCV infection include serological tests to detect HCV antibodies and core antigen, molecular tests to detect and quantify HCV RNA, and genotyping techniques. Serological Assays NS2/3 Protease PKRBD: Protein Kinase Resource Binding Domain 2209-PSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEI-2274 ISDR: Interferon Sensitivity Determining Region HCV screening and initial diagnosis are usually determined according to clinical symptoms and elevated liver enzymes (especially alanine aminotransferase [ALT]), and positive enzyme immunoassay (EIA) or chemiluminescence immunoassay (CIA) for anti-hcv antibodies in patients with known risk factors (Figure 2). First- (EIA-1), second- (EIA-2), and third- (EIA-3) generation enzyme immunoassays detect antibodies against a variety of HCV core, NS3, NS4, and NS5 antigens. Currently, second- and third-generation EIAs NS3/4 Serine Protease Figure 1 Physical map of hepatitis C virus (HCV) genome and function of structural and nonstructural proteins. Ser-prot indicates serine protease; NTPase, RNA nucleoside triphosphatases, IFN, interferon. are the principal laboratory tests used to detect HCV exposure. Seropositivity by these tests occurs as early as 8 to 10 weeks after exposure to the virus and the tests remain positive for 6 months to a lifetime after infection (Figure 2). The specificity of EIA-2 and EIA-3 is 99% or greater. 14 The sensitivities of EIA-2 and EIA-3 are 95% and 97%, respectively, in high-prevalence populations. 15 The anti-hcv EIA screening kits approved by the United States Food and Drug Administration (FDA) and used in United States laboratories include the Abbott HCV EIA 2.0 and AxSYM HCV 2.0 (Abbott Laboratories, Abbott Park, IL), and ORTHO HCV Version 3.0 ELISA (Ortho-Clinical Diagnostics, Inc., Raritan, NJ). The Abbott HCV EIA 3.0, AxSYM HCV 3.0, and IMx HCV 3.0 (Abbott Laboratories) used for the initial screening and detection of antibodies against a variety of recombinant HCV core, NS3, NS4, and NS5 antigens are not currently approved by the FDA. FDA-approved CIA assays include the VITROS Anti-HCV assay for use in the VITROS ECi/ECiQ Immunodiagnostic System (Ortho-Clinical Diagnostics, Inc), and recently approved Architect Anti-HCV (Abbott Laboratories), ADVIA Centaur (Siemens AG, Munich, Germany), Elecsys Anti- HCV Immunoassay (F. Hoffman-La Roche Ltd., Basel, Switzerland), and OraQuick HCV Rapid Antibody Test (OraSure Technologies, Inc., Bethlehem, PA) methods. The latter is a CLIA-waived strip immunoblot assay used Fall 2013 Volume 44, Number 4 Lab Medicine Review_Amjad.indd 293

3 Figure 2 Pathologic and serologic response to acute and chronic hepatitis C virus (HCV) infection. ALT indicates alanine aminotransferase; HCV, hepatitis C virus. Titer RNA ALT RNA RNA HCV Core Antigen Anti-HCV Antibody Table 1. Hepatitis C Virus Antibody Detection Assays for Screening and Diagnosis Anti-HCV Antibody Assay Method HCV Antigens Screening Type(s) Abbott HCV EIA 2.0 a EIA Core, NS3, NS4 Initial, donor ADVIA Centaur b CIA for ADVIA Centaur Core, NS3, NS4, NS5 Initial Architect Anti-HCV a CIA for Architect Core, NS3, NS4 Initial AxSYM HCV 2.0 a EIA for AxSYM Core, NS3, NS4, NS5 Initial Elecsys Anti-HCV Immunoassay c CIA for Cobas e411 Core, NS3, NS4 Initial ORTHO HCV Version 3.0 ELISA d EIA Core, NS3, NS4, NS5 Initial, donor VITROS Anti-HCV Assay d CIA for ECi/ECiQ Core, NS3, NS4, NS5 Initial Abbott HCV EIA 3.0 a EIA Core, NS3, NS4, NS5 Initial IMx HCV 3.0 a EIA for IMx Core, NS3, NS4, NS5 Initial EIA: enzyme immunoassay, NS, nonstructural; CIA: chemiluminescent Immunoassay. a Abbott Laboratories, Abbott Park, IL. b Siemens AG, Munich, Germany. c F. Hoffman-La Roche Ltd., Basel, Switzerland. d Ortho-Clinical Diagnostics, Raritan, NJ. primarily for screening and point-of-care testing. The specific features of these assays are outlined in Table 1. In a population at low risk for HCV infection (eg, individuals who do not use intravenous drugs, have no history of transfusion, human immunodeficiency virus [HIV] infection, end-stage renal disease, or hemodialysis, or do not have multiple sexual partners), a negative EIA or CIA result is sufficient to rule out infection. Patients with HIV or endstage renal disease undergoing hemodialysis may have false negative anti-hcv antibody test results. All patients with a positive HCV EIA or CIA result, and high-risk patients with negative EIA or CIA results and unexplained elevated ALT levels should be further tested via nucleic acid tests (NAT) to detect the presence of HCV RNA Months Symptoms ± Symptoms ± Years distinguish a true HCV antibody positive from a false positive with a second HCV antibody assay approved by FDA for the diagnosis of HCV infection, and that is different from the assay used for the initial antibody testing. 16 Nucleic acid tests (Table 2) can be used as supplemental tests along with the anti-hcv antibody assays. Detection of HCV RNA is helpful in certain situations; for example, the presence of HCV RNA in the absence of anti-hcv antibodies indicates acute infection, which can be confirmed by seroconversion a few days to a few weeks later. If HCV RNA is positive in a patient with a positive screening test result, HCV RNA detection has the advantage of detecting the presence of active HCV infection as well as verifying the presence of anti-hcv antibodies. 14 According to the most recent CDC recommendations, supplemental HCV antibody tests can be used to Finally, according to CDC recommendations, intial testing for HCV infection needs to be performed by a rapid or 294 Lab Medicine Fall 2013 Volume 44, Number Review_Amjad.indd 294

4 Table 2. HCV RNA Detection Assays for Diagnosis and Monitoring of Therapy a Tests for Diagnosis and Management Sensitivity or of HCV Infection Method Range (IU/mL) Use(s) Qualitative Cobas Amplicor HCV, version 2.0 b RT-PCR 50 Confirmation of diagnosis, determination of rapid and sustained virological response VERSANT HCV RNA c TMA 5 Screening, confirmation of diagnosis Quantitative Abbott Real-Time HCV d Real-time RT-PCR Management of HCV-infected patients during treatment COBAS AmpliPrep/COBAS TaqMan HCV Test b Real-time RT-PCR Management of HCV-infected patients during treatment COBAS TaqMan HCV version 2.0, for use with Real-time RT-PCR Management of HCV-infected patients during treatment the HighPure System b VERSANT HCV RNA 3.0 c bdna Management of HCV-infected patients during treatment HCV, hepatitis C virus; RT-PCR, reverse transcriptase polymerase chain reaction; TMA, transcription-mediated amplification; bdna, branched-chain DNA. a All assays mentioned in this table have been approved by the United States Food and Drug Administration. b F. Hoffman-La Roche Ltd., Basel, Switzerland. c Siemens AG, Munich, Germany. d Abbott Laboratories, Abbott Park, IL. laboratory-conducted assay to detect the presence of HCV antibodies. A nonreactive HCV antibody result indicates no HCV detected. A reactive result indicates one of the following: 1) current HCV infection, 2) past HCV infection that has resolved, or 3) false positivity. A reactive result should be followed by NAT for HCV RNA. If HCV RNA is detected, that indicates current HCV infection. If HCV is not detected, that indicates either past, resolved HCV infection, or false HCV antibody positivity. 16 Nucleic acid-based RT-PCR is necessary to detect and quantify HCV RNA and to confirm positive results on EIA and CIA testing of patient samples. HCV RNA measurement by nucleic acid amplification is also essential for determining past chronic and resolved HCV infection. An alternative to nucleic acid tests is determination of the HCV core antigen via serological methods. The HCV core antigen is detectable 1 to 2 days after HCV RNA becomes detectable. HCV core antigen kinetics parallel HCV RNA and thus can be used as a marker of HCV replication. The HCV core antigen assay is independent of genotype and has a specificity of 99.5%. The limit of detection is 1.5 pg/ ml, which corresponds to an HCV RNA concentration of approximately 10,000 to 50,000 IU/mL. 17 Commercially available assays include the Architect HCV Ag assay (Abbott Laboratories), which comprises 5 different antibodies to detect HCV core antigen, is highly specific (ie, 99.8%), and has sensitivity for determination of chronic hepatitis C equivalent to that of HCV RNA measurement. 18 The detection limit of the HCV antigen assay corresponds to HCV RNA concentrations of 600 to 1000 IU/mL. 17 The clinical usefulness of this test is in question, given the availability of sensitive nucleic acid amplification tests and the fact that most HCV treatment algorithms are guided by qualitative and quantitative HCV RNA PCR results. Molecular Tests to Detect and Quantify HCV Determination of chronic HCV infection status, treatment decisions, and monitoring of treatment response depend on 3 important factors: detection of HCV RNA, quantification of RNA, and determination of HCV genotype. Several commercial and lab-developed nucleic acid based assays are currently available to detect, quantify, and genotype HCV (Table 2). These assays vary with regard to their ranges of detection, sensitivities, specificities, cost, and ease of use. HCV RNA can be detected in a patient s blood within 1 to 3 weeks after infection and at the onset of symptoms (Figure 2). However, in some patients, as the anti- HCV antibody titer increases, the HCV RNA level can decrease. Thus, HCV RNA may not be detectable during the acute phase of the infection; however, this finding is rare and usually transient, and if chronic infection develops, HCV RNA PCR test results usually become positive. HCV antibodies are not protective and do not Fall 2013 Volume 44, Number 4 Lab Medicine Review_Amjad.indd 295

5 prevent development of chronic HCV infection. Repeated absence of HCV RNA in the presence of HCV antibodies in test results indicates resolved infection. 14 Nucleic acid amplification tests detect the presence of HCV RNA using a combination of amplification and detection techniques. Nucleic acid amplification tests are classified as qualitative, namely, qualitative polymerase chain reaction (PCR), transcription-mediated amplification (TMA), or quantitative, namely, branched-chain DNA (bdna) amplification and quantitative real-time PCR. 19 Whereas qualitative test results for HCV RNA are sufficient to confirm diagnosis, quantitative results are used to monitor treatment. The most commonly used commercially available qualitative tests are the reverse-transcriptase PCR (RT- PCR) based AMPLICOR 2.0 HCV test and the semiautomated COBAS AMPLICOR 2.0 (F. Hoffman-La Roche Ltd., Basel, Switzerland). These tests have a lower limit of detection, namely, less than 50 IU/mL, and have specificity between 96% and 99%. 19 VERSANT HCV RNA (Siemens AG, Munich, Germany), another FDA-approved qualitative assay, is based on TMA using an isothermal nucleic acid sequence-based amplification procedure in which target RNA is amplified without a reverse-transcription step. TMA is a more sensitive test, with a lower limit of detection of 5 IU/mL and specificity of 99.5%. 20,21 Qualitative assays are less expensive than quantitative assays and are used to confirm EIA results and monitor the virologic response to antiviral treatment. Measurement of HCV RNA is essential for making treatment and management decisions. 22 Quantitative assays are used at different time points before, during, and after treatment to indicate whether HCV is present or has been eliminated, and to determine when and whether treatment should be stopped or continued. HCV RNA can be quantified by target gene amplification techniques (competitive or real-time RT-PCR), or by signal amplification techniques (bdna assays). Among the quantitative RT-PCR assays are the COBAS AmpliPrep/COBAS TaqMan HCV Test, and COBAS TaqMan HCV for High Pure system (F. Hoffman-La Roche Ltd), as well as the Abbott Real-Time HCV assay (Abbott Laboratories). The Abbott Real-Time HCV assay is an in vitro RT-PCR assay for use with the Abbott msample preparation system and with the Abbott m2000sp and m2000rt instruments for the quantitation of HCV RNA in human serum or plasma. The VERSANT HCV RNA, version 3.0, (Siemens AG), also FDA approved, is a bdna signal amplification assay that does not require thermal cycling. These commercially available assays have specificities between 98% and 99% and can detect HCV RNA over a wide range of concentrations, from low levels of approximately 10 IU/ml to as high as 10 million IU/ml (Table 2). These assays are used in the management of HCV infected patients and are used to monitor the viral load in response to the antiviral treatment. Genotyping Techniques HCV genotyping is performed at baseline to identify candidates for treatment and to select appropriate therapies. HCV genotypes can be determined by several methods that target core, E1, NS4, and NS5 viral proteins, as well as the 5'UTR regions of the HCV genome. HCV genotyping methods include PCR amplification of the target gene and sequencing, PCR amplification and hybridization with genotype-specific probes, and real-time RT-PCR. 23 Currently, there are no FDA-approved assays to determine HCV genotypes. The reference method for HCV genotyping is PCR amplification and direct sequencing of HCV genomic NS5B or 5'UTR regions, alignments, and phylogenetic analysis. 8,9 These methods are time consuming, require techniques and equipment usually used in the research laboratory and not in the clinical laboratory, and mostly are used in epidemiological studies in which exact genotyping is needed. However, the advantage of direct sequencing is that it reveals genomic variability and the presence of quasi-species during the natural progression of disease and its response to antiviral therapy. 23 In clinical practice, HCV genotyping is performed using commercially available kits that employ PCR amplification and hybridization with genotype-specific probes. Currently used methods include reverse-hybridization line probe INNO-LiPA HCV II assay (Innogenetics, Ghent, Belgium), simplified direct sequencing Trugene 5'NC HCV Genotyping assays (Siemens AG), and the Abbott Real-Time HCV Genotype II Assay (Abbott Laboratories) (Table 3). These assays have a high degree of concordance and reliability. Incorrect typing of the major genotypes is rare (<3%); mixed genotypes occur but are uncommon. Occasionally (ie, <5%), specimens cannot be genotyped. This usually results from low viral levels, difficulty with the PCR amplification, or extreme nucleotide variability within the HCV genome. 24,25 INNO-LiPA HCV II is a reverse-hybridization line probe assay that uses specific oligonucleotide probes to capture 5' URT of the HCV genome. The Versant HCV Genotyping Assay (INNO- LiPA), version 2.0 (comparable to the earlier INNO-LiPA) 296 Lab Medicine Fall 2013 Volume 44, Number Review_Amjad.indd 296

6 Table 3. HCV Genotyping Assays Genotyping Technique Genomic Region/Method Specificity/Use(s) INNO-LiPA HCV II a 5' UTR/line probe Identify genotypes 1-6; difficult to subtype 1 and 6 Versant HCV Genotyping Assay (INNO-LiPA) 2.0 b 5' UTR, core/line probe Identify genotypes 1-6; better distinction of subtypes 1c, 6 and subtypes 1a and 1b; difficult to type 2 and 4 Trugene 5'NC HCV Genotyping b 5' UTR/sequencing Difficulties in subtyping; NS5B sequence under development Abbot Real-Time HCV Genotype II Assay c 5' UTR, NS5B Genotyping 1a (NS5b), 1b (NS5b), 2a, 2b, 3, 4, 5, 6 (5 UTR) HCV, hepatitis C virus; UTR, untranslated region; NS, nonstructural. a Innogenetics, Ghent, Belgium. b Siemens AG, Munich, Germany. c Abbott Laboratories, Abbott Park, IL. is a next-generation line probe assay that detects the 5' URT and the core region of the HCV genome. Currently, the INNO-LiPA HCV, version 2.0 (Siemens AG), is the most effective commercial assay for HCV genotype 1 subtype identification and is often used in clinical trials and practice. Addition of core region identification allows better subtyping of genotype 1 and characterization of genotype In the earlier version of the INNO-LiPA genotyping assay, amplicons from COBAS AMPLICOR HCV, version 2.0 (F. Hoffman-La Roche Ltd.), can be used. However, in the newer VERSANT HCV Genotyping Assay, version 2.0 (comparable to the earlier INNO-LiPA), which can also test the core region, HCV viral RNA needs to be amplified using the VERSANT HCV amplification kit, version 2.0 (Siemens AG). 27 The Trugene 5'C HCV Genotyping Kit (Siemens AG, Munich, Germany) is based on the sequence analysis of 5' UTR and comparison with the contents of genomic libraries. 28 The Abbott Real- Time HCV Genotype II Assay (Abbott Laboratories) is a real-time PCR-based method that quantifies HCV RNA and can determine HCV 1a, 1b, 2a, 2b, 3, 4, 5, and 6 genotypes. Resistance to Interferon and Monitoring of Treatment HCV genotypes and subtypes play an important role in treatment response and patient outcome. Patients infected with genotypes 2 and 3 are more likely to respond to therapy, compared with genotype 1 infection. Until recently, the conventional treatment of chronic HCV infection was pegylated interferon alpha in combination with ribavirin for 24 weeks in patients with HCV genotypes 2 or 3, and for 48 weeks in patients with genotype 1. Recently, the FDA approved 2 HCV NS3/4A protease inhibitors for use in patients infected with genotype 1 HCV. These new drugs include VICTRELIS (boceprevir; Merck & Co., Inc., Whitehouse Station, NJ) and INCIVEK (telaprevir; Vertex Pharmaceuticals Inc., Cambridge, MA), and are used in combination with pegylated interferon alpha and ribavirin. This regimen is becoming the current standard of care in this group of HCV 1 infected patients. Patients with other genotypes continue to be treated with a combination of pegylated interferon and ribavirin. Successful treatment is defined as a sustained virologic response (SVR) in which there is continued undetectable viral load at 24 weeks after completion of treatment. In two studies, SVR was achieved in 42% to 51% of patients with genotype 1 (average, 50%) and 73% to 82% of patients with genotype 2 and 3 infection (average, 77%) with the pegylatedinterferon and-ribavirin regimen. 29,30 The genotype-1 response rates have improved to approximately 75% to 80% with addition of the new protease inhibitors. Numerous studies have been undertaken to explain resistance to interferon therapy in patients infected with HCV genotype 1. These studies have implicated the role of envelope 2 (E2) protein, a non-structural (NS)3/4A serine protease, and NS5A proteins as possible mediators of interferon resistance. The role of NS5A has been emphasized because it contains a protein kinase resource binding domain (PKRBD), that includes an interferon sensitivity-determining region (ISDR) and a nuclear localization signal (NLS) and V3 region (Figure 1). The results of these studies indicate a relationship between the number of mutations in the ISDR regions of the HCV gene and response to interferon treatment. 34,35 Initially, these observations were not confirmed by studies in Europe and the United States. However, statistical analysis of many published sequences of ISDR from HCV genotype 1b isolates has demonstrated a strong relationship between response to interferon treatment Fall 2013 Volume 44, Number 4 Lab Medicine Review_Amjad.indd 297

7 and the number of mutations in the ISDR. 36,37 Therefore, NS5A ISDR mutations are of great interest as a possible cause of HCV resistance to interferon therapy. However, the benefit of the ISDR sequence as a prognostic guide for patients with HCV infection receiving interferon therapy has not been established. A genetic test to detect the presence of a single polymorphism within the human interleukin-28b (IL-28B) gene has been reported to help predict patient response to therapy. The importance of the IL28B polymorphism was first reported in Since then, several studies have indicated that specific variations in this gene are associated with a 2- to 3-fold greater risk of sustained viral suppression in response to treatment with combination pegylated interferon alpha/ ribavirin therapy among patients infected with genotype 1 HCV Based on these observations, a patient may be identified as an IL28B responder; this type has a better chance of spontaneous clearance or successful treatment, compared to non-responder genotypes. This genetic test to detect the presence of a single polymorphism within the IL28B gene may help patients predict a patient s response to therapy Conclusion The use of serological and molecular methods has become essential in the diagnosis, management, and treatment of patients infected with HCV. Quantitative RT-PCR assists the monitoring of treatment and determination of the response to antiviral therapy. 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