An Overview of the Laboratory Assay Systems and Reactives Used in the Diagnosis of Hepatitis C Virus (HCV) Infections

In the mid-1970s, a new disease entity termed ‘non-A, non-B’ (NANB) hepatitis was first described and, in the following years, led to discovery of the causative virus, posttransfusion, and to community-acquired NANB hepatitis increasingly becoming recognized as a potentially serious disease that results in liver cirrhosis and/or hepatocellular carcinoma.16, 26 Hepatitis C virus (HCV) was first identified in 1989 using molecular methods at the Chiron Corporation, but to date, the virus has never been visualized or grown in cell culture.37 Hepatitis C virus (HCV) is a single-stranded RNA virus with a genome of about 10 000 nucleotides containing a single large, continuous open reading frame and with organization most closely resembling the Flaviviridae.11 HCV is a global healthcare problem and the World Health Organization (WHO) estimates that at least 170 million people (3 % of the world’s population) are infected with HCV worldwide and most of the patients are concentrated in developing countries. 48

Trends in Immunolabelled and Related Techniques 340 Fig. 1.Organization of the HCV genome and polyprotein. 26

Classification of laboratory assay systems and reactives used in the diagnosis of hepatitis C virus infection.
Immunoasssay systems used in the diagnosis of hepatitis C virus infections may be divided into four groups.

HCV RNA assay.
Diagnostic tests used for the diagnosis of HCV infections may be divided into three groups according to aim of use. 22Screening test (Anti-HCV tests based on EIA or CIA) 2. Supplemental test (Strip immunoblot assay-SIA) 3. Confirmatory test (HCV RNA) (HCV Core Ag as a pre-confirmative test.).
Detection or quantification of HCV RNA an important molecular assay based on the principle of target amplification used for confirming test results of immunoassay reactives.Reactives used in order to diagnose HCV infections also may be divided in to two groups according to identification method of the test. 32n Overview of the Laboratory Assay Systems and Reactives Used in the Diagnosis of Hepatitis C Virus (HCV) Infections 341 2. Direct tests: (HCV RNA, HCV Ag, HCV genotyping assays and sequencing of HCV genome).

Anti-HCV assay
Diagnostic procedures of hepatitis C virus infection in laboratories are principally based on the detection of antibodies (IgG) against recombinant HCV polypeptides by two main methods: Enzyme immunoassay (EIA) and Chemiluminescence immunoassay (CIA).The anti-HCV assay is used as a screening test.Non-structural and recombinant antigens have been used in the production of immunoassay reactives.Serologic and virologic markers of past or present HCV infection include IgG antibodies (anti-HCV).An assay for Ig M anti-HCV is available, but it does not distinguish between acute and chronic HCV infection. 25ree different generations of anti-HCV test kits have been developed to date.The firstgeneration HCV enzyme immunoassay (EIA) detected only antibodies against the nonstructural region 4 (NS4) with recombinant antigen c100-3. 25With the development of secondgeneration tests, additional antigens from the core region (c22-3), the NS3 region (c33c) and a part of c100-3 (named 5-1-1) from the NS4 region were used. 17The third-generation EIA anti-HCV test used today includes an additional antigen from the NS5 region and a reconfiguration of the core and NS3 antigens. 46Anti-HCV assays have several disadvantages, such as a high rate of false positivity, a lack of sensitivity of detection in the early window periods of 45 to 68 days after infection, the inability to distinguish between acute (ongoing active, viremic), past (recovered) and persistent (chronic) infections and a possibility of false negativity with samples from immmunocompromised patients who may not have an adequate antibody response. 19,30,33,36 Inrder to shorten the duration of the diagnosis of heaptitis C virus infection especially in preseroconversion period being capable of the detection of antibodies against NS 5 proteins means that a third generation reactive is very important for anti-HCV assays.
Because there remains a window period, estimated at 82 days with the second-generation assays, at 66 days with the third generation assays, between the infection and the detection of HCV antibodies 12 .NS5, enables the detection of HCV antibodies on an average of 26 days earlier in individuals with transfusion-transmitted HCV infection.Furthermore, sensitivity is also improved (approaching 97%), specifically in a high-prevalence population. 14mmarized properties of the fully automated CIA based systems and anti-HCV reacitves shown in Table 1.

Strip immunblot assay (SIA)
SIA rectives used as supplementary test, involving recombinant immunblot assay (RIBA) HCV 3.0 strip immunoassay (SIA) (Chiron Corporation, Emeryville, CA, USA), which contained recombinant antigens (c33c, NS5) and synthetic peptides (5-1-1, c100 and c22); and INNO-LIA™ HCV Score (Innogenetics N. V. Ghent, Belgium) is a 3rd generation line immunoassay which incorporates HCV antigens derived from the core region, the E2 hypervariable region (HVR), the NS3 helicase region, the NS4A, NS4B, and NS5A regions.Both SIA's are based on the principle of an enzyme immunoassay.Recombinant immunoblot assays are used as supplementary test of anti-HCV assays.As types of EIA, recombinant immunoblot assays also have several disadvantages, such as being difficult to perform, a high percentage of 'indeterminate' results and a high cost.Therefore, these anti-HCV assays are not often used in developing countries or in routine diagnostic laboratory procedures.

Hepatitis C virus core antigen (HCV Ag) assay
Total serum HCV core antigen, a surrogate marker of HCV replication, can also be detected and quantified.A commercial assay kit for it is available.HCV core antigen can be detected on average, 1 to 2 days after HCV RNA during the pre-seroconversion period.HCV Ag test is presented as a ''Direct marker for diagnosis of HCV infection''.Sensitivity of HCV core antigen test is slightly lower than HCV RNA assay but many studies carried out with HCV c o r e A g t

e s t c o m p a r e d w i t h H C V R N A , p r o v e d t h a t t h e H C V A g t e s t i s s p e c i f i c , reproducible, highly sensitive and clinically applicable assay. HCV antigen test also
showing good correlation comparing with HCV RNA.HCV core antigen test may be used as a second line confirmatory test alternative to HCV RNA. 23,28,29 I is also needed as a preconfirmatory test for anti-HCV results and to distinguish false positives from the accurate ones.This is because it is easy to perform, reliable, has a high specificity and sensitivity rate, is cost effective, is able to shorten the duration of the diagnosis of patients during the window period and has a lower risk of laboratory contamination than assays based on nucleic acid amplification technology. 45,50 ring the past decade, several HCV Ag tests have been developed as potential alternatives to HCV RNA assay. 5The first was developed by Tanaka et al. 45 1995, and then Aeyogi et al. 1 developed a new and 100-fold more sensitive test.In previous studies, the HCV Ag was detected one day after the HCV RNA in patients undergoing seroconversion. 12,13,34 Te Architect HCV Ag assay (Abbott Laboratories, Diagnostics Division, Abbot Park, IL, USA) is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective and applicable as a screening, and pre-confirming test for anti-HCV assays in the laboratory procedures used for the diagnosis of hepatitis C virus infection.The Architect HCV Ag assay was performed using the automated Architect ®

Molecular diagnostic systems, and reactives used for the HCV RNA and HCV genotyping assays
Confirmation test is needed.Although third-generation HCV reactives are more sensitive and specific than older generation assays, they still have a high percentage of false positive reactions, so that it is mandatory to confirm every reactivity, especially with low titers, by anti-HCV CIA or EIA with HCV RNA assay (reactives with a lower limit of detection of 50 IU/mL or less) to avoid false positive results.To minimize the likelihood of false-positive anti-HCV results, the CDC has recommended the confirmation of all anti-HCV results by either RIBA or HCV RNA assay. 28,29  RNA is the earliest marker of infection, and a direct indicator of ongoing viral replication.It appears 1 to 2 weeks after infection before any alterations in liver enzyme levels and appearance of anti-HCV antibodies can be detected.If the nucleic acid testing (NAT) result is positive, active HCV infection is confirmed.If NAT result is negative, the HCV antibody or infection status can not be determined.NAT assays are used to detect and quantify HCV RNA. 8,18,40 HRNA assay systems can be divided into two groups: qualitative and quantitative HCV RNA.
Qualitative HCV RNA assay.Target amplification methods used qualitative detection of HCV RNA and have lower limits of detection of 5-50 IU/mL: Reverse Transcriptase-PCR (single enzyme RT-PCR, dual enzyme RT-PCR, nested RT-PCR), TMA (transcriptionmediated amplification), NASBA (isothermal RNA amplification).This group includes the qualitative RT-PCR, of which the Amplicor™ HCV 2.0 (Roche Molecular Systems, USA) is an FDA-and CE-approved RT-PCR system for qualitative HCV RNA testing that allows detection of HCV RNA concentrations down to 50 IU/ml of all HCV genotypes. 31ranscription-mediated amplification-(TMA)-based qualitative HCV RNA detection has a very high sensitivity (lower limit of detection 5-10 IU/ml). 20,41 ranscription Mediated Amplification (TMA) Component System, Versant™ HCV RNA Qualitative Assay (Siemens Healthcare Diagnostics, Germany) is also commercially available which is accredited by FDA and CE and provides an extremely high sensitivity, superior to RT-PCR-based qualitative HCV RNA detection assays. 21,42,43 Qtitative HCV RNA assay.The Abbott RealTime™ HCV Test provides a lower limit of detection of 12 IU/mL, a specificity of more than 99.5 % and a linear amplification range from 12 to 10,000,000 IU/mL independent of the HCV genotype. 27,39 ERSANT kPCR Molecular System Siemens Healthcare Diagnostics is also avialable as a real time PCR system for quantification of HCV RNA.Rotor Gene Q real time PCR device and Qiagen HCV RNA kits (Qiagen GmbH, Germany) are used for quantification of HCV RNA by real time PCR method with specificity of 99.0 %, a lower limit of detection 34 IU/ml and capable to detect up to 10, 000,000 IU/mL.
In certain situations HCV RNA result can be negative in persons with active HCV infection.
As the titre of anti-HCV increases during acute infection, the level of circulating HCV RNA declines; intermittent HCV RNA positivity has been observed among persons with chronic HCV infection.A negative HCV RNA result can also indicate resolved infection.Follow-up HCV RNA testing is indicated only in persons with serologically confirmed anti-HCV positive results. 8,18 ection and quantification of HCV RNA is used as the only one confirmative test of all the anti-HCV, HCV Ag assays and SIA tests.The HCV RNA assay is a reliable method but needs technical skill and may also result in false positivity because of contamination.It is time consuming and expensive. 18,38 CV RNA is extensively used to confirm antibodybased screening test results.Amplification methods (target amplification by RT-PCR, transmission-mediated amplification (TMA), and signal amplification by b-DNA-branch-DNA are the most expensive methods (45-50 USD per test for real-time PCR, 10-12 USD per test for HCV Ag CIA, and 5-6 USD per test for anti-HCV CIA) when compared with anti-HCV and HCV Ag tests; and require sophisticated technical equipment and highly trained personnel.One specific problem with the HCV RNA assay is that HCV RNA can be temporarily undetectable because of the transient, partial control of viral replication by the immune response.Patients in a period of non-viremia may be detected as anti-HCV-positive and HCV RNA-negative.In such a situation, the HCV RNA test should be repeated a few weeks later with a new sample.This need for re-testing is a disadvantage of the HCV RNA test.In addition, nucleic acid amplifications are time-consuming methods and have the risk of laboratory contamination.for these reasons, amplification methods are not suitable for wide use in most laboratories, especially in developing countries. 2,10,24,38,44 HCVnotyping assay.HCV has six genotypes represented by digits (1-6) and multiple subtypes represented by letters (a, b, c…) and most recently a seventh HCV genotype have been characterized.HCV genotyping should be carried out in every patient before antiviral therapy. 6Both direct sequence analysis and reverse hybridisation technology allow HCV genotyping.Reverse-hybridization method and kits (The VERSANT HCV Genotype Assay (LiPA Line Prob Assay) 2.0, Bayer HealthCare LLC, Tarrytown, NY, USA) also exist for hepatitis C virus genotype assay.The test is mainly based on biotinylated DNA, generated by RT-PCR amplification of the 5'untranslated region (5'UTR) of HCV RNA, is hybiridized to immobilized oligonucleotide probes.The VersantTM HCV Genotype 2.0 System (Siemens Healthcare Diagnostics) is suitable for indentifying genotypes 1-6 and more than 15 different subtypes and is currently the assay for HCV genotyping.By simultaneous analyses of the 5'UTR and core region, a high specificity is achieved especially to differentiate the genotype 1 subtypes (1a versus1b).The TruGene direct sequence assay determines the HCV genotype and subtype by direct analysis of the nucleotide sequence of the 5'UTR region.Incorrect genotyping rarely occurs with this assay.However, the accuracy of subtyping is poor.The current Abbott RealTime™ HCV Genotype II assay is based on real-time PCR technology, which is less time-consuming than direct sequencing.Preliminary data reveal a 96% concordance at the genotype level and a 93% concordance on the genotype 1 subtype level when compared to direct sequencing of the NS5B and 5'UTR regions.
Interpretation of HCV and acute hepatitis C test results are presented in Tables 2 and 3.

False positivity problem and reasons of false positive results of anti-HCV immunoassay reactives
Although the present third generation EIA tests have better sensitivity and specifity rates than their predecessors, there still exists a high prevelance of false-positive results, especially among low risk group, immunocompromised patients or populations without liver diseases, leading to unnecessary cost-effective health expenditures and confusing diagnostic challenges.The most common problem in the laboratory screening assay of anti-HCV is the false positivity of low titers. 3,7 ng immunocompromised populations (e.g., hemodialysis patients) the average rate of false-positive results is approximately: 15 %.False positive anti-HCV results obtained from both CIA and EIA-based reactives can be explained by the fact that no structural antigens and proteins have been derived from HCV up to now.HCV has not been cultured and natural viral proteins are not available.Confirmatory test should be used in order to discriminate the false positive results from the accurate ones. 49

Reasons of false results of anti-HCV immunoassay reactives.
The amino acid sequence and the purity of the HCV antigen used for assay development are significant factors influencing both the specifity and the sensitivity of anti-HCV immunoassays.Because of the high IgG concentration in human blood (>5 mg/ml)-e.g. in paraproteinemia or auto-antibody production or after Ig G denaturation caused by repeated freezing and thawing or by heat-inactivation of serum samples , there is a strong tendency for some of the IgG molecules to be bound to the micro-well surface by direct adsorbtion or by indirect capture via the surface molecules, and then arouse a signal, giving false-positive results.This problem might be more serious when the samples are from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), portal cirrhosis, and some infectious diseases due to the very complicated, higher concentration of immunoglobulin components in their blood. 49

Table 1 .
Summarized properties of CLIA based basic assay systems and reactives.
29,38SR CIA system (Abbott Laboratories, Diagnostics Division, Abbot Park, IL, USA).The Architect HCV Ag assay is a two-step chemiluminescent microparticle immunoassay technology for the quantification of the HCV Ag in human serum or plasma samples.The sample volume required is 110 µl, and the total assay time is 36-40 min.The cutoff value is 3.00 fmol/liter (0.06 pg/mL); thus, values <3.00 fmol/l are considered nonreactive, values ≥3.00 fmol/l are considered reactive and values ≥3.00 fmol/l and <10.00 fmol/l are retested in duplicate.If both retest values are nonreactive, the specimen is considered nonreactive for HCV Ag.If one or both of the duplicates have a value ≥3.00 fmol/l, the specimen is considered repeatedly reactive.29,38 9,47tification of HCV RNA can be determined by target amplification techniques Real-Time PCR assays), or by signal amplification methods (branched DNA-bDNA Assay).Several FDA-and CE-approved standardised systems are commercially available.The Cobas Amplicor™ HCV Monitor (Roche Diagnostics) is based on a competitive PCR technique whereas the Versant™ 440 HCV RNA Assay (Siemens Healthcare Diagnostics) is based on a bDNA technique.Both have restricted lower limits of detection (500-615 IU/ml).More recently, the Cobas TaqMan assay and the Abbott RealTime™ HCV test, both based on real-time PCR technology, have been introduced and now replaced the qualitative and quantitative methods.All commercially available HCV RNA assays are calibrated to the WHO standard based on HCV genotype 1.It has been shown that results may vary significantly between assays with different HCV genotypes despite standardisation.9,47

Table 2 .
26terpretation of HCV test results.26Third-generationEIA,2HCVRNA assay with a lower limit of detection ≤50 IU/mL.*Generally seen in patients who have recovered from a past HCV infection.RIBA should be used.A positive RIBA with two or more HCV-RNA positive results suggest that HCV infection resolved.A negative RIBA result indicates the false positivity of the EIA result, in the both no further testing is needed.**Acutely infected patients can also have HCV RNA and anti-HCV at the time of diagnosis.It is difficult in these cases to distinguish acute hepatitis C from an acute exacerbation of chronic hepatitis or acute hepatitis of another cause in a patient with chronic hepatitis C. 1

Table 3 .
8nterpretation of acute hepatitis C test results.8