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Indian Journal for the Practising Doctor

Evolutionary Changes in Diagnosis of Hepatitis C: A Brief Review of Laboratory Diagnosis

Author(s): Irshad M, Kawatra M

Vol. 5, No. 6 (2009-01 - 2009-02)

Irshad M, Kawatra M

Dr. M. Irshad (Professor) and Dr. Mallika Kawatra (Sr. Resident), Clinical Biochemistry Division, Department of Laboratory Medicine, All India Institute of Medical Sciences (AIIMS), New Delhi

Correspondence: Prof. M. Irshad, Clinical Biochemistry Division, Department of Laboratory Medicine, A.I.I.M.S., New Delhi-110029, INDIA. [Ph. No.: 011-91-26594981; Fax No.: 011-91-26558641; E-mail: drirshad54(at)yahoo.com]

ISSN: 0973-516X

Abstract

This brief report describes the sequential modifications and improvements made in the development of various assays for laboratory diagnosis of hepatitis C virus (HCV) infection after its characterization in 1989. The first assay developed was the ‘first generation ELISA’, followed by assays with further improvement in the sensitivity and specificity, labeled subsequently as ELISA-2 and ELISA-3. The generation of ‘recombinant immunoblot assay’ (RIBA) was to facilitate confirmation of the cases detected anti-HCV positive by ELISA. Both types of assays detected anti-HCV antibodies in serum. HCV molecular assays, such as detection of HCV-RNA by reverse transcription and polymerase chain reaction, were developed to detect live virus particles and for use in following therapeutic efficacy. Quasispecies detection, genotyping and serotyping were done to unravel the genetic diversity in HCV variants so that therapeutic strategies could be planned more accurately. All these assays have been described in a sequence of their need and place in diagnosis.

Keywords: HCV, ELISA, RIBA, RT-PCR, Genotype

Introduction

The hepatitis C virus (HCV) is a positivestranded RNA virus that was first characterized in 1989 by Choo et al.1 It was soon identified as the main causative agent of the previously called posttransfusion, non-A non-B hepatitis.2 The virus has a positive sense, ssRNA genome approximately 10 kb in length, with similarities in genome organization and some sequence homology with pestiviruses and flaviviruses.3,4,5 Different isolates of HCV show substantial nucleotide sequence variability distributed throughout the viral genome.6 Regions encoding the putative envelope proteins [(E1, E2 / non-structural protein 1 (NS-1)] are the most variable,7 whereas the 5` non-coding region (5`NCR) is the most conserved.8,9 Comparison of the published sequences of HCV has led to the identification of a number of distinct virus type that may differ from each other by more than 30% over the whole viral genome.3 Till now, six major HCV- genotypes and almost 80 subtypes have been reported.

HCV infection is reported to be the most common blood-borne infection in the USA and Europe. It is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Much effort has been directed towards establishing therapeutic strategies against the HCV genome. Since the response of anti-viral therapy against HCV depends on the type of HCV variant, so, in addition to its detection, genotyping also has become important in routine diagnostic laboratories. Accurate diagnosis is essential to identify the presence of HCV-RNA in apparently symptom free carriers or to monitor the viral load of treated patients. Present review briefly describes the historical changes occurring in the diagnosis of HCV infection with current status of its diagnostic tools used in different laboratories.

Screening of HCV infection

Before HCV was characterized in 1989, screening of blood-borne non-A, non-B infection was done with aminotransferases, particularly ALT estimation in serum, a surrogate marker for non-A, non-B infection. ALT level raised more than 1.5 times of normal value was taken as the possible indication of this infection in patients at risk of blood-transmitted diseases. After the virus was characterized, an enzyme-linked immunosorbent assay (ELISA) was developed for detecting anti- HCV antibodies in serum. This was the first generation ELISA and incorporated the 100-3 epitope from the non-structural NS4 region of HCV genome. This ELISA was commercially available and was widely used world over. Since then three generations of serodiagnostic anti-HCV tests have been developed, each new generation providing additional improvement in sensitivity to anti-HCV antibodies. In fact, the problem of false positivity as well as detection of lately produced or pre-existing antibodies with first and second generation ELISA, prompted the researchers to further improve the assay system. The third generations ELISA was designed to detect antibodies to four recombinant HCV proteins encoded by core NS3, NS4 and NS5 regions of HCV genome.

The third-generation assay differs from the second-generation ELISA by substitution of the NS5 protein for the 5-1-1 antigen used in second generation assay. However, this substitution does not appear to account for the increased sensitivity of the newer assay.10 The average period for HCV seroconversion after blood transfusion has been shortened with each successive generation; it got reduced to 7 to 8 weeks with ELISA-3 compared to 10 weeks with ELISA-2 and 16 weeks with ELISA- 1.10

ELISA assays have many advantages in the diagnostic setting including ease of automation, ease of use, relative cost-effectiveness, low variability and high sensitivity in screening. Some of the major disadvantages include suboptimal sensitivity and specificity, an abundance of false-positives in lowprevalence populations and poor sensitivity in postliver transplant patients because of immunosuppression.11

Supplemental or confirmatory tests for HCV

An accurate diagnosis of hepatitis C virus (HCV) is necessary before treatment and counseling of patients begins. As stated above, false-positive results following ELISA testing continue to be noted among low-risk blood donors. While elevated aminotransferases and high-risk factors for infection are indicative of active infection and hepatitis, additional testing for antibody specificity can further document HCV infection. A number of confirmatory and/or supplemental serodiagnostic tests are available to cross-check seropositive results obtained with ELISA screening tests. An HCV diagnosis can be confirmed by the recombinant immunoblot assay (RIBA), which identifies antibodies to individual HCV antigens and has a higher specificity than ELISA.12 Confirmation can involve the use of either a four-antigen RIBA (RIBA HCV 2.0) or strip immunoblot assay (SIA). The second-generation RIBA, or RIBA-2, uses the same recombinant antigens as the ELISA-2. A more sensitive third generation RIBA has also been introduced for use.

RIBAs are technically more demanding than ELISA. Also, RIBA positivity is not always a true indicator of active infection by HCV because recovered patients may stay anti-HCV positive for years. Conversely, RIBAs are simpler, more standardized, and more reproducible. The thirdgeneration RIBA has resolved many of the RIBA-2 interdeterminate samples. However, only 50% of the RIBA-3 positive blood donors are HCV RNA positive by polymerase chain reaction (PCR) assay12,13 which is the next step in HCV diagnosis. RIBA 3 is a confirmatory assay and exposes several specific HCV peptides from two nonoverlapping core regions (C1 and C2), E2, NS4 and NS5A regions, and recombinant NS3 of the HCV polyprotein. The HCV nonstructural 5A (NS5A) protein may contribute to the interferon-resistant phenotype of HCV.

Molecular assays: detection and quantization

While the diagnosis of HCV is currently based on the detection of antibodies by ELISA or RIBA, the technique is less sensitive in the early phases of HCV infection and cannot differentiate between active infection and disease resolution. Also, immunocompromised patients such as those who are infected with HIV, or hemodialysis patients, produce fewer antibodies.14 The direct molecular qualitative detection of HCV RNA by reverse transcription (RT) and PCR are considered the gold standard for the diagnosis of HCV infection and for assessing the antiviral response to interferon (INF) therapy.

Quantitative assessment of HCV RNA levels, via signal amplification and quantitative PCR (Q-PCR), are valuable tools in the clinical management of patients before, during and after therapy. PCR-based assays are able to ascertain minute amounts of HCV RNA in serum or plasma. HCV RNA detection by PCR helps to resolve weakly positive or negative ELISA results when clinical signs and/or risk factors are compatible with HCV infection.

A reliable, standardized assay for HCV RNA can convey 1) whether a patient is likely to respond to INF therapy and 2) if a virologic response has occurred, besides 3) promoting a better comprehension of the relationship between viral load and the natural history of chronic HCV infection.15 In studies by Davis et al16 and McHutchison et al,17 HCV RNA levels correlated with rates of response to INF and/or ribavirin therapy.

Another assay, the branched chain DNA assay (bDNA), based on a signal amplification, is highly standardized. The second generation bDNA provides a modest increase in sensitivity compared to the previous generation and minimal bias in measuring HCV RNA levels for the major HCV genotypes. Direct detection of as many as 1,000 hepatitis viral genomes is possible.18

The clinical value of bDNA assay has been the object of several performance studies. Jacob et al15 compared the relative sensitivities of first-and second generation branched nucleotide assays.

Moreover, comparing the bDNA with a semiquantitative cDNA-polymerase chain reaction (cDNA-PCR) in monitoring HCV RNA levels, the bDNA assay was not as sensitive as cDNA-PCR, given its user friendliness and quantitative results, but it is considered a useful test for monitoring HCV RNA levels in patients treated with INF. However, patients who are non-reactive in the bDNA assay have to be re-tested by cDNA-PCR because low viral titers are not detected by the bDNA assay.19

Sequence determination and phylogenetic analysis

Sequencing of the E2 HVR1, followed by phylogenetic analysis is recommended for studying patient-to-patient transmission, analysis of interspousal transmission, nosocomial infections in a haemodialysis unit and in geographical regions with a high endemicity of only one subtype. In a multivariate analysis of pretreatment parameters with a sustained virological response to treatment, three parameters appeared to be independent predictors of a treatment response: a low viral load, a low anti- HCV core IgM titre, and a low genetic complexity of HVR1 major variants.20 However, the E2 HVR is too heterogeneous to be of value for classification of HCV genotypes. Instead, the 5’ NCR, core, E1 and NS5B regions are frequently amplified for the purpose of genotypic classification.

Phylogenetically analyzing a subtype can point to the possibility of mutation and reversions. Nucleotide sequence analysis of hepatitis C virus (HCV) strains showed substantial variability leading to a classification into several genotypes and subtypes. The data correlating HCV genotypes and subtypes with hepatitis C viremia levels, host characteristics (age, mode of transmission, duration of infection), and severity of liver disease conflict. The interpretation of clinical studies is further complicated because the molecular methods used lacked specificity for genotyping/subtyping and underestimated viremia levels, especially in patients infected with HCV genotypes 2 and 3.

Genotyping/Subtyping: Divergence within the human population

Not only do HCV quasispecies sequences express variability in different regions of the genomes, but isolates also differ among themselves. All isolates separate into phylogenetically-related clusters called subtypes. One or several subtypes can be classified into several major types that show similarities over 65-75% of the total genome. The term genotype is used generically to refer to subtypes, types or both. Use of the term genotype to describe quasispecies variants is not appropriate. Eleven HCV genomes are known to exist21 as well as more than 90 subtypes,22 with more subtypes being discovered at a continuous rate. 21

Several screening tests have been developed to identify HCV genotype, and include reverse hybridization line probe assay ‘restriction fragment length polymorphism’ (RFLP) of the PCR amplicons, and nested PCR with genotype-specific parameters to the core region.

The optimal genotyping region is reported to be the 5’ untranslated region (UR) because of high conservation within, but considerable variation between, genotypes. In the technique based on the reverse hybridization principle, in that biotinylated PCR fragments are hybridized to a selection of highly specific immobilzed probes. In a second step, the biotin group in the hybridization complex is exposed by incubation with a streptavidin-alkaline phosphatase complex and the appropriate chromogen compounds. Such a technique allows discrimination of HCV types and subtypes and is capable of detecting single nucleotide differences in the 5’ UR.

In a more recent study, 21 probes dispersed over seven variable 5’ UR areas were applied to hybridization technique and used to analyze HCVinfected sera from different geographical regions resulting in an abundance of subtypes. The investigators concluded that the selected probes detected the corresponding signature motifs in the seven variable regions with 100% reliability.22 In addition, these motifs allowed correct type interpretation of samples collected worldwide. Finally, subtyping specificities vary according to geographical region, with 11 prototype subtyping patterns identifying the majority of samples from Europe and the Americas. These results indicate that such a reliable assay applicable to routine typing and subtyping of HCV specimens.23

In RFLP analysis, a single PCR fragment is amplified from a certain region of the HCV genome with universal primers. Restriction enzyme recognition sites present in the DNA fragment usually show subtype- or type-specific distribution. Thus, restriction fragments with varying lengths are created after cutting the PCR fragment with one or several restriction endonucleases. The electrophoretic separation of these fragments lets the observer infer the approximate lengths of the restricted fragments and, in turn, identify the genotype.

Serotyping

Determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules for patients with chronic hepatitis C. The suitability of two versions of a serological, so-called serotyping assay based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes, were studied by Pawlotsky.24 The NS4, E1 and a small variable region in the core yield type-specific antigenic determinants. Type-specific B-cell epitopes have also been reported in the NS4A and NS4B regions, thus, single or branched peptides obtained from the NS4A and NS4B regions can be used for serotyping. For serological determination of HCV genotype in the study, serotyping assays were compared to reverse hybridization probe assay. The results showed that these assays remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivity between different HCV genotypes could be responsible for the mistyping.

Conclusions

Tests used in the diagnosis and management of HCV infection have several advantages and disadvantages. ELISAs have many advantages in the diagnostic setting including ease of automation, ease of use, relative cost-effectiveness, low variability and high sensitivity in screening. The false-positives that they can produce can be cleared through the use of RIBAs. Taken one step further, measurement of HCV RNA by reverse transcription or PCR can actually plot virological response to INF. Detecting and quantifying quasispecies can explain why a particular patient is unable to clear an HCV infection or has become INF-resistant as well as explain why an isolate-specific vaccine is not effective. The genetic, geographical and etiological variations in the hepatitis C virus are observable via genotyping, allowing for more exact diagnosis. Genotyping has also important implications for development of hepatitis-related vaccines and biotherapeutic agents.

Acknowledgement: The authors thank and appreciate the financial aid provided by the Indian Council of Medical Research, New Delhi (India) to conduct this study. Authors are also thankful to Mrs. Suman Rawat for preparing this manuscript.

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