Sunday, July 24, 2011

. Laboratory Diagnosis of Hepatitis C Virus Infection.

Due to the lack of typical symptoms diagnosis of acute hepatitis C is rarely established. Because HCV antibodies may develop later during the course of hepatitis C diagnosis of acute infection is based on the detection of HCV-RNA together with a history of contact to a HCV positive source, a course of increased liver enzymes from previous normal levels and/or the absence of HCV antibodies (Jaeckel et al.,  2001 and Gerlach et al., 2003). 
            After screening for chronic HCV infection by anti HCV antibody testing past or ongoing hepatitis C in anti-HCV positive patients is determined on the basis of HCV core antigen and HCV-RNA. During selection of patients for screening of hepatitis C one has to be aware that typical symptoms of chronic liver damage and even elevated liver enzymes are often not present in patients with HCV infection. This together with a relative slow progression of the disease has led to the fact that at present only in a minority of patients diagnosis of  CHC is established.  After proof of CHC the need for antiviral therapy has to be assigned on the basis of the level of liver enzymes, histological grading and staging of liver damage, extra hepatic manifestations of the disease, as well as the social and personal situation of the patient (National  Institutes of  Health, 2002).
             For management of treatment, determination of HCV genotype provides important information about the chance of sustained virologic response and the projected duration of interferon alfa-based antiviral therapy (Hadziyannis et al., 2004 and Zeuzem et al., 2004).
             The key parameter for assessment of antiviral response during therapy is HCV-RNA. Precise and reliable quantification and highly sensitive detection of HCV-RNA before, during and after interferon alfa based antiviral therapy is critical for determination of virologic response and premature discontinuation of therapy in virologic-non-responders (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003 and Davis et al., 2003). Detection of HCV antibodies.
            The first enzyme immunoassay was introduced in 1989/90 and was able to detect antibodies to a c100–3 named antigen, a recombinant protein from the non-structural (NS)4 region of the HCV genome (Ortho Diagnostic Systems Inc., Raritan, NJ, USA) (Kuo et al., 1989).
            Today’s third-generation enzyme immunoassays (EIA-3) are available since 1993 and were completed with recombinant proteins of the NS5 region. In comparison with the second generation assays (detection of core, NS3 and NS4 proteins) a 2–3 weeks earlier detection of HCV antibodies was shown and specificity to detect HCV antibodies in blood donors was improved from 99.90% to 99.95% (Uyttendaele et al., 1994 and Barrera et al., 1995). Sensitivities in patients with chronic liver disease and in panels of sera were estimated at 98.90% and 97.20%, respectively (Colin et al., 2001). Generally, results for anti-HCV screening are only reported as positive or negative. Differentiation of acute and chronic hepatitis C may be possible by quantification of anti HCV core IgG levels. IgG antibodies levels typically are higher in chronic compared to acute hepatitis C and usually persist in immunocompetent patients (Nikolaeva et al., 2002).  After spontaneous recovery from acute hepatitis C or successful eradication of the virus, disappearance of anti-HCV IgG below the detection limit of the assays may be seen after many years (Wiese et al., 2000). Detection of HCV antigens.
            After screening for HCV infection with HCV antibody tests confirmation of ongoing hepatitis C is performed by the detection of HCV core antigen and HCV-RNA. For routine purposes HCV antigens will be detected in the blood while for scientific research liver tissue or lymphocytes may be used alternatively. After acute infection HCV-RNA will become detectable within the first 2 weeks and during chronic infection virtually all patients are HCV-RNA positive using sensitive assays. Due to a lower sensitivity the use of HCV core antigen tests instead of HCV-RNA assays with some restrictions will lead to the same results. During chronic hepatitis C, HCV core antigen and HCV-RNA assays are used for monitoring of antiviral therapy for prediction and confirmation of virologic response, before, during and after treatment (Bouvier-Alias et al., 2002 and Veillon et al., 2003). HCV core antigen.
            The introduction of a reliable and sensitive HCV core antigen assay was hampered by different difficulties: (i) the development of specific monoclonal antibodies recognizing all different HCV subtypes; and (ii) the need of accumulation and dissociation of HCV particles from immune complexes for increasing sensitivity. Recently, a first HCV antigen detection system was approved by the legal authorities and has become commercially available in the US and Europe (Ortho, trak-CTM, Ortho Clinical Diagnostics, Raritan, NJ, USA). In this assay, after dissociation of HCV particles from immune complexes and lysis, HCV core proteins are bound to coated monoclonal antibodies in a microwell. Following several washing steps a bound core antigen is incubated with an anticore-specific Fab antibody fragment conjugated with horseradish peroxidase. After a second wash quantitative detection is performed by the addition of ophenylenediamine (OPD)/hydrogen peroxide and measurement of the optical density. HCV core concentrations are calculated against a curve obtained from standards and are expressed as picograms per milliliter.  The limit of detection established by the manufacturer is 1.5 pg/mL. The HCV core antigen assay is highly specific (99.5%), genotype independent, and has shown a low inter- and intra-assay variability (coefficient of variation 5–9%) (Bouvier-Alias et al., 2002 and Veillon et al., 2003).
            After infection with HCV detection of HCV core antigen is delayed approx. 1–2 days after HCV-RNA becomes detectable (Icardi et al., 2001 and Cividini et al., 2003).
            As for HCV-RNA, no correlation between levels of HCV core antigen and elevation of liver enzymes or the grade of inflammation and the stage of fibrosis in the liver was observed. From the results of different studies it was shown that the lower detection limit of 1.5 pg/mL for core antigen equals to approximately 10,000–50,000 IU/ml (Bouvier-Alias et al., 2002 and Veillon et al., 2003). 
            In a study with 139 HCV antibody and HCV-RNA positive patients presented in an outpatient clinic six (4%) were HCV core antigen negative.  In these patients HCV-RNA concentrations were measured between 1,300 and 58,000 IU/mL highlighting the limitations to use the HCV core antigen assay for confirmation of ongoing hepatitis C in HCV antibody positive patients (Veillon et al., 2003).
            Comparison of HCV core antigen and HCV-RNA quantification show an excellent correlation (correlation coefficient, r =0.92). One pg/mL of HCV core antigen equals to 8,000 IU/mL HCV-RNA with a intersubject variation from 5,000 to 12,000 IU/mL (Bouvier-Alias et al., 2002 and Veillon et al., 2003)  Studies for evaluation of the utility of HCV core antigen concentrations for prediction of virologic response/non-response before and during interferon-based antiviral therapy are currently under way. Preliminary data of a small study investigating 38 patients treated with peginterferon-alfa and ribavirin observed a potential use of a positive HCV core antigen test (>1.5 pg/mL) for discontinuation at week 12 with a high negative prediction value (100%non-response) (Buti et al., 2004). 
            However, as shown by others, due to the limited sensitivity transient negative results by the HCV core antigen assay were associated with continuously positive HCV-RNA concentrations. The aim of determination of early virologic response certainly is to discontinue as many virologic non-responders as possible from further treatment with an ideal 100% prediction value. Thus, the proportion of nonresponder patients to be discontinued from further treatment at week 12 and 24 by core antigen and HCV-RNA measurement, respectively, has to be carefully assessed in large cohorts of patients (Rebucci et al., 2003). Detection of HCV-RNA. Qualitative assays (RT-PCR, TMA).
            After detection of HCV in 1989 the first test systems to confirm ongoing, replicating hepatitis C were based on RT-PCR. Due to a high conservation of the 5/ nontranslated region (NTR) of the HCV genome throughout the different geno- and subtypes, primers complementary to this region were chosen for reliable diagnostics (Christoph, 2004).
             Due to their lower detection limits in comparison with quantitative HCV-RNA assays, qualitative HCV-RNA tests clinically are used for diagnosis of acute hepatitis C in which HCV-RNA concentrations are fluctuating and may be very low and for confirmation of virologic response during, at the end and after antiviral therapy (Christoph, 2004). 
            By the end of 1993 a first standardized RT-PCR based assay for detection of HCV-RNA was introduced (AmplicorTM HCV, Roche Molecular Systems, Pleasanton, CA, USA). The Amplicor™ HCV system is a combined single tube-, single enzyme-, single primer set RT-PCR assay (Christoph, 2004). 
            More recently, a second test system based on transcription mediated amplification (TMA) was approved for qualitative detection of HCV-RNA (VersantTM Qualitative HCV, Bayer Diagnostic, Emeryville, CA, USA). HCV-RNA detection by this technique consists of threesteps which are performed in a single tube: (i) target capture; (ii) target amplification and; (iii) specific detectionof target amplicons by hybridization protection assay.
            Due to its extreme high sensitivity the TMA-based assay (lower detection 5–10 IU/mL) is able to detectresidual HCV-RNA amounts not observed by standard RT-PCR-based tests (lower detection limit 50 IU/mL). In 7–33% of patients treated with (pegylated) interferon-alfa and ribavirin with negative results by RT-PCR at the end of treatment residual HCV-RNA  concentrations were detectable by TMA (Sarrazin  et al., 2000; Comanor et al., 2001 and Sarrazin  et al., 2001).
            Similar results were obtained during therapy at week 24 for decisions of treatment discontinuation in HCV-RNA positive patients. Whether patients with low HCV-RNA levels (TMA positive/RT-PCR negative) will benefit from prolongation of therapy is still unknown (Mihm et al., 2004). Quantitative assays (RT-PCR, real time RT-PCR bDNA)
            Measurement of HCV-RNA concentration in serum/blood is an important parameter for management of chronic hepatitis C. While no correlation was observed between the HCV-RNA viral load and the severity or the progression of liver disease HCV-RNA concentration is an important predictor for response to antiviral therapy. In multiple studies an inverse correlation of lower pretreatment HCV-RNA blood levels with higher rates of sustained virologic response to (pegylated) interferon alfa combination therapy with ribavirin was observed. Furthermore, after initiating interferon-based therapy the initial decline of HCV-RNA levels is used for determination of virologic non-response at week 12 and week 24 of treatment. Patients without an initial decline of HCV-RNA of at least 2 log steps and/or absolute values above 30,000 IU/mL have shown to become virologic non-responders in 98–100% of cases (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            Thus, according to current recommendations treatment should be discontinued on the basis of this week 12 HCV-RNA decline rule. Highly sensitive HCV-RNA detection assays (lower detection limits ≤50 IU/mL) are used for determination of virologic response at week 24 of therapy. As patients with detectable HCV-RNA at this time point will not achieve a sustained virologic response in 98–100% of cases again treatment can be discontinued at week 24 on the basis of a positive highly sensitive HCV- RNA test (Fig. 4) (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
Figure 4 . Management of (pegylated) interferon alfa/ribavirin combination therapy (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            Figure (5) gives an overview on commercially available qualitative and quantitative HCV-RNA detection assays. Different techniques are used for quantification of HCV-RNA: (i) assays with HCV-RNA (target) amplification on the basis of standard or real time PCR; and (ii) a branched DNA (signal) amplification method without multiplication of the original amount of HCV-RNA in the probe (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            Five different quantitative HCV-RNA detection assays are approved by the legal authorities and are commercially available. Three are based on standard RT-PCR (SuperquantTM, National Genetics Institute, Los Angeles, CA, USA; Cobas AmplicorTM HCV Monitor version 2, Roche Molecular Systems, Pleasanton, CA, USA; LCxTM HCV-RNA Quantitative, Abbott Laboratories, North Chicago, IL, USA) and one is based on branched DNA technique (VersantTM Quantitative HCV-RNA, bDNA 3.0, Bayer Diagnostics, Emeryville, CA, USA). Recently, the first real-time PCR based assay for HCV-RNA quantification was approved (COBAS TaqManTM , Roche Molecular Systems, Pleasanton, CA, USA) and in the future additional real-time PCR based HCV quantification assays may follow as this technique has the potential of highly sensitive, linear quantification of RNA and DNA targets (Cristoph, 2004).

Figure 5. Characteristics of qualitative and quantitative HCV RNA detection assays. Numbers below the balls/columns represent lower detection limits of the assays. (●) qualitative assays; (■) real time PCR-based linear quantitative assays; (▓) standard PCR-based non-linear quantitative assays; (░ ) signal amplification bDNA-based linear quantitative assay (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            HCV-RNA extraction from blood samples before detection with the HCV-RNA quantification assays is performed by different techniques (i.e. standard lysis and precipitation, glass fiber filter columns, hybridization to magnetic particles). To yield lower detection limits between 10 and 50 IU/mL higher sample volumes are needed for extraction of HCV-RNA. While for the real-time PCR-based Cobas TaqManTM assay as well as for the TMA-based assay 500mL are used for HCV RNA preparation, nucleic acids are extracted from 100mL and 50mL in the Cobas AmplicorTM Monitor and bDNA 3.0 assays, respectively. Automation of nucleic acid extraction requires a further increase with samples volumes up to 1 mL (Christoph, 2004).
            Lower and upper detection limits of linear amplification of HCV-RNA together with intra and interassay variabilities of the different assays are summarized in (Table 1). Taken together, precision data of the different standard PCR assays are comparable. The signal amplification based bDNA assay showed a slightly lower variability. The slightly higher variability of the real-time PCR based Cobas TaqManTM assay is explained by the extreme wide range of linear amplification from 35 to 7 Mill. IU/mL. Together with HCV-RNA preparation by the glass fiber filter-based HPSTM system the Cobas TaqMan assay at present is restricted for the use of quantification of HCV genotypes 1 and 6. For the remaining HCV genotypes (2, 3, 4, 5) underestimation of HCV-RNA concentrations were observed. The reason for this underestimation so far is unknown and studies to resolve the problem are currently under the way(Yu et al., 1999; Ross et al., 2002; Leckie et al., 2004 and Sarrazin et al., 2004).
            All commercially available HCV-RNA quantitative assays are standardized against the WHO HCV international standard (96/790) and titer results are automatically reported in international units (IU/mL). For calculation of results of previous versions of the different assays given in copies individual conversion factors to IU exist (Table 2) (Pawlotsky et al., 2000). While during long-term storage a significant decrease of HCV-RNA levels may be observed (Schmid et al., 1999), storage at room temperature during the first 72–96 h after blood collection was not associated with a decline of HCV-RNA concentrations (Grant  et al., 2000 and Kessler et al., 2001).

HCV RNA quantification assay



mean SD

mean SD

Amplicor MonitorTM HCV, version 2.0


5 × 105

0.080 log10

0.063 log10

LCxTM HCV Quantitative


2.3 ×106

0.075 log10

0.066 log10

SuperquantTM HCV





Cobas TaqManTM HCV


7 × 106

0.088 log10

0.103 log10

VersantTM Quantitative HCV, version 3.0


8 × 106

0.050 log10

0.030 log10

Table 1. Characteristics of commercial HCV-RNA quantitative assays *Data obtained from (Yu et al., 1999; Ross et al., 2002; Leckie et al., 2004 and Sarrazin et al., 2004). LDL, lower detection limit; UDL, upper detection limit; Inter-assay, inter-assay variability; Intra-assay, intra-assay variability; n.a., data not available.
Table 2. Conversion factors from copies to IU (Pawlotsky et al., 2000).

HCV RNA quantification assay

Conversion factor

Amplicor Monitor HCV, version 2.0

1 IU = 2.5 copies/mL

LCx HCV Quantitative

1 IU = 4.3 copies/mL

Superquant HCV

1 IU = 3.4 copies/mL

Versant Quantitative HCV,version 3.0

1 IU = 5.2 copies/mL

2.2.11. Prevention.
            The development of an effective HCV vaccines remains an unsolved challenge. This is due to the lack of suitable cell culture system and small animal models to establish HCV infection for evaluating crucial virus neutralizing pathways. During the last decad, many efforts have been dedicated to the design of either prophylactic or therapeutic HCV vaccines, but still with little success (Forns et al., 2002).
            Passive protection using high-titer specific immunoglobulin is also currently unavariable. At present the role of specific HCV neutralizing antibodies in the prevention and control of infection has not been defiend clearly, prevention of HCV transmission can be achieved only by preventing exposure to the virus. HCV transmission can be minimized by educating intravenous drug users not to reuse or share syringes and needles or any other items involved in drug taking, simple techniques for sterilizing such equipment have been suggested for preventing HCV transmission in intravenous drug users. Organ donors should be screened for evidence of HCV infection before transplantation, also transmission through sexual contact can be minimized by protected sex. Many different approaches have been currently tested using structural (E1-E2-Core) and non structural HCV protein as candidates for different strategies of immunization (Choo et al., 1994; Geissler et al., 1997 and Forns et al., 2002). New adjuvants are also evaluated to improve immunogenicity and to favor the generation of a strong cellular response (Sfolander and Cox, 1998).

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