Abstract

This review mainly focuses on research findings in the area of Hepatitis C virus (HCV) infection, diagnosis, and treatment which is based on published literature and covers the different aspects of HCV. HCV infection previously called as blood transmitted non-A, non-B infection and is prevalent globally and poses a serious public health problem worldwide. The diagnosis of HCV infection includes non-specific anti-HCV antibodies detection by using OraQuick® HCV Rapid Antibody Test kit as a tool for initial screening of individuals having symptoms of HCV infection or for those who are at risk. Upon positive results, an FDA (Food and Drug Administration) approved confirmatory HCV RNA assay called nucleic acid testing (NAT) is performed as a supplemental testing.

 Detection of viral nucleic acid in serum using polymerase chain reaction (PCR) technique is very common among all viral infections. Current PCR assays detect viral nucleic acid with high accuracy and the exact copy number of viral particles. Moreover, multiplex assays using real-time PCR are available for identification of HCV-genotypes and their subtypes. In contrast to previous methods, the newly developed assays such as FDA approved PCR-based tests for qualitative detection of HCV RNA (Amplicor Hepatitis C Virus Test and Cobas Amplicor Hepatitis C Virus Test) are not only fast and economic, but also resolve the many problems related to differentiation between present and past infection.

Sofosbuvir is a highly potent inhibitor of the NS5B polymerase in HCV and showed high efficacy in combination with several other drugs in the treatment of Hepatitis C infection for different genotypes. However, recently it is reported in 2016 that sofosbuvir leads to significant metabolic changes such as glucose and cholesterol metabolism may be significantly altered.

1. Introduction

HCV infection is a major health problem worldwide. The geographical distribution of HCV infection varies from place to place. North Africa, East Asia, and the Middle East have the highest prevalence of HCV (Negro and Alberti, 2011). A recent study based on anti-HCV seroprevalence data estimated that 185 million people, nearly 2.8% of the world's population, have been infected with HCV (Hanafiah et al., 2013). The Centers for Disease Control and Prevention (CDC) estimates that in the US alone approximately 29,700 new cases are diagnosed per year, a number that is steadily increasing. The hepatitis C infection may lead to chronic hepatitis, decompensated cirrhosis, and hepatocellular carcinoma, causing high mortality rate which is approximately 700,000 individuals per year (Goossens et al., 2016).

1.1. Discovery of HCV

In 1970 Harvey J. Alter and his colleagues reported a huge number of hepatitis infection cases that were caused due to blood transfusion and it was also proved that all of these cases were due to neither hepatitis A nor hepatitis B viruses (Feinstone et al., 1975). These cases of hepatitis were thus called non-A, non-B hepatitis (NANBH) for more than 10 years. The causative agent responsible for spreading of NANBH was hepatitis C virus (HCV). HCV was first isolated and identified in 1989, after extensive screening of bacterial clones derived from experimentally infected chimpanzee samples by researchers from the Chiron Corporation in California. Since the isolation of HCV, the interest in this field has expanded remarkably and a growing knowledge of the virus life cycle has led to the development of potent drugs (Choo et al., 1989; Houghton, 2009).

1.2. Genotypes of HCV

Analysis of the nucleotide sequence homology of different viral genomic regions such as the 5′ noncoding (5′NC) region and regions coding for the envelope glycoprotein (E1), core, and nonstructural 5B (NS5B) proteins led to the identification of 7 different genotypes and approximately 67 subtypes within these genotypes (Smith et al., 2014). HCV circulates within a single patient as closely related variants named quasispecies. This constant variation of the HCV genome is the main problem faced in the development of a vaccine against HCV. On the basis of nucleotide homology analysis of the non-structural 5B (NS5B) regions of the HCV genome, it has been estimated that strains from different genotypes have 67 – 69% similarity between them. Within subtypes of HCV only 20–25% of nucleotides are different (Simmonds et al., 2005).

Although genotype does not predict the outcome of infection but it predicts treatment response against a particular genotype and may help to determine the duration for cure. Therefore, genotyping can be performed by using restriction fragment length polymorphism (RFLP) technique (Davidson et al,. 1995), by direct sequence analysis or by reverse hybridization to genotype-specific oligonucleotide probes.

1.3. Prevalence and Geological Distribution of HCV Genotypes

Recently, Messina et al., (2015) demonstrated that genotype 1 is the most predominant (42%), followed by genotype 3 (30%). The sum of genotype 2, 4, and 6 corresponds to approximately 23%, while genotype 5 represents less than 1% of the total number of HCV cases. HCV genotype 7 was first described in 2014 (Smith et al., 2014) and has been reported so far in only a few patients. Genotype 1 and 3 are widely spread throughout the world. However, the prevalence of genotype 3 is particularly high in South Asia whereas other genotypes have more restricted geographical distributions such as genotype 2 predominates in West Africa, genotype 4 in the Middle East, genotype 5 in South Africa, and genotype 6 in East and South East Asia. The prevalence of genotypes and subtypes is different due to its dependence on the transmission route. For example, genotypes 1a and 3a are more frequent among intravenous drug users while genotype 1b has a high prevalence among patients who were infected through blood transfusions (Pawlotsky et al., 1995).

1.4. Transmission of HCV

The principal route of transmission of HCV is via the blood such as syringe exchange between drug users, transfusion of unscreened blood especially among those patients who are receiving long-term dialysis, tattoos, piercing, reuse or improper sterilization of contaminated medical equipment, and needle-stick injuries. In developed countries, since the systematic screening of blood donors and the extensive implementation of safety procedures, Hepatitis C infection risk after transfusion or organ transplant has dramatically decreased. Mother-to-baby transmission can also occur (especially if the mother is co-infected with HIV). HCV can be transmitted sexually, however, this route of transmission is uncommon. In groups of people with high-risk sexual behavior, such as HIV-positive men who have sex with men, the incidence of HCV is higher than in the general population and has increased in recent years (Wandeler et al., 2012).

2.      Structure and Life Cycle of HCV

The hepatitis C virus (HCV) belongs to the Hepacivirus genus of the Flaviviridae family of viruses. HCV is an enveloped, positive-strand RNA virus that is spherical and has a diameter of between 40 and 80 nm in HCV-infected patients (Bradley et al., 1985). It is found in blood in the form of lipo-viro-particle (LVP) which is composed of an envelope mainly lipoproteins (derived from host cell membranes), two viral glycoproteins, envelope proteins E1 and E2, and an icosahedral capsid containing a positive-sense, single-stranded RNA (ssRNA) genome as shown in Figure 2.1. The length of HCV ssRNA molecule is 9.6 kb (Choo et al., 1991), flanked by 5’ and 3’ untranslated regions (UTR). The genomic ssRNA contains two open reading frames (ORF). The large ORF encodes the entire HCV polyprotein of ~3000 amino acids which undergoes a series of cleavages to form several type structural and non-structural proteins (Figure 2.2). The structural proteins are cleaved from the polyprotein by cellular signal peptidases within

the endoplasmic reticulum and nonstructural proteins by virally encoded proteases. All the structural proteins have hydrophobic C termini. This feature is thought to be important for membrane association and cleavage from the polyprotein by host signal peptidases (Xu et al., 2001).

 The 5’UTR is highly conserved among different HCV isolates and the secondary structure contains four distinct stem-loops called internal ribosome entry sites (IRES) that are essential for the cap-independent translation of the genomic RNA (Fraser and Doudna, 2007). The 3’UTR contains a variable untranslated region of approximately 30 nucleotides downstream of the 3' terminus of the polyprotein-coding region followed by a poly-U/UC and 3’X region. The extreme 3' terminus is a highly conserved region of 98 nucleotides, known as 3’X region which forms three stem-loop structures. Although functional role of 3’UTR region is unclear, however, a recent study demonstrated that 3’UTR may enhance translation by transferring the host translation machinery components from the 3’ to the 5’ end of viral RNA (Bai et al., 2013).

Once virus releases its RNA strand inside the host cell, it is translated into a single polyprotein of about 3,000 amino acids. This large molecule is then cleaved by proteases into several domains: three structural proteins (C, E1, and E2), a small protein called p7, and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). These nonstructural proteins help in the viral replication (Bartenschlager & Lohmann, 2000).

3.      Diagnosis for HCV

Late diagnosis of the HCV infection is partly due to the asymptomatic initial phase of the disease and limits the potential benefits of antiviral drugs. In clinical practice the usual approach to test HCV infection, begins with HCV antibody initially for antibodies to HCV (anti-HCV), then to use HCV ribonucleic acid (RNA) to perform qualitative assays such as copy number detection and qualitative assays such as genotyping of Hepatitis C virus. A nonreactive HCV antibody result indicates no HCV antibody detected. A reactive result should be followed by nucleic acid test or NAT for HCV RNA confirmation as a supplemental test. If HCV RNA is detected, that indicates current HCV infection. If HCV RNA is not detected that indicates current HCV infection, past resolved HCV infection or false HCV antibody positivity (Khuroo, 2015).

3.1. Serological Assays

The diagnostic procedures for hepatitis C virus infection used in laboratories are based on the detection of antibodies to Hepatitis C virus (anti-HCV) against recombinant HCV proteins using enzyme immunoassay (EIA) and chemiluminescence immunoassay. Non-structural and recombinant antigens are used in these assays. Modern EIAs detect antibodies that bind to recombinant antigens derived from four viral regions: core, NS3, NS4, and NS5. The serological assays are divided into initial HCV antibody assays and nucleic acid testing (NAT) assay (Irshad et al., 2013).

3.1.1 Testing for HCV Antibodies

The OraQuick® HCV Rapid Antibody Test kit method was approved in 2010 by FDA and used as an initial screening to detect a patient’s exposure to the Hepatitis C virus. The presence of the antibodies associated with HCV (anti-HCV antibodies) can help us to determine if a person is currently infected or has previously been infected with HCV.

The OraQuick® HCV Rapid Antibody Test is performed by taking a blood sample from a person with signs or symptoms of HCV infection or at a risk of having HCV infection. This blood sample is added to the solution vial containing the developing solution. Then the test strip coated with HCV antigens is placed into the vial. If there are antibodies to the HCV in the blood, they stick to the test strip and react with the chemicals and a colored line appears within 20–40 minutes on the test strip in the result window. This line indicates the presence of anti-HCV antibodies in the patient’s blood and the outcome is reported as reactive. A positive test result should be confirmed with a supplemental test as false positive results may occur. It is relatively less expensive, gives quick results and more precise than other screening methods.

3.1.2 Testing for HCV RNA

If testing is desired to distinguish between true positivity and biological false positivity for HCV antibody, then, a second assay named nucleic acid testing or NAT may be performed for the detection of HCV RNA in serum or plasma of the blood of an individual whose test was reactive for HCV antibodies. NAT for HCV was first introduced in Germany in 1997. It is different from the assay used for initial antibody testing because it is based on amplification of targeted regions of viral ribonucleic acid and detects the genetic material of the virus itself, rather than signs of the body's immune response to the infection. It is highly sensitive and specific for viral nucleic acids due to its ability to detect as few as a single copy of the HCV genetic material in a sample and detects them earlier than the other screening methods (Hans & Marwaha, 2014).

3.2. Qualitative Assays

HCV replicates at relatively low levels, meaning that HCV RNA cannot be detected in body fluids by means of classical hybridization techniques and that can be detected in the blood using target amplification techniques such as transcription-mediated amplification (TMA) or polymerase chain reaction (PCR) (Martell et al., 1999).

3.2.1 Transcription-Mediated Amplification (TMA)

Transcription-mediated amplification (TMA) is considered one of the most sensitive detection assays for hepatitis C virus RNA in serum. It is the patented single-tube nucleic acid amplification technology in which only two enzymes are used to drive the reaction: RNA polymerase and reverse transcriptase. This technique was developed by Hologic, that allows Procleix NAT solutions to perform every step of the screening process in a single tube, with fewer material transfer steps and less processing time. Hence, it reduces the risk of cross-contamination in the laboratory and deliver results faster than other amplification technologies.

The first step, after RNA extraction, involves the conversion of the target portions of the HCV RNA into complementary DNA (cDNA) with the help of reverse transcriptase. Then RNA polymerase initiates transcription and synthesizes single-stranded RNA (Martell et al., 1999). Some of the newly synthesized ssRNA amplification products re-enter the process and serve as templates for new rounds of amplification and billions of copies are generated in less than one hour. Acridinium ester (AE)-labeled probes specifically hybridize to these amplified products and detect them. There are some important advantages of TMA which include; amplification of multiple targets, faster time to results, and exponential amplification of only desired pathogen targets (Comanor et al., 2001).

Real-time target amplification techniques are also used for qualitative assays of HCV RNA. In real-time PCR, each round of amplification leads to the emission of a fluorescent signal which can be detected in the closed tube during the reaction. The FDA has approved two PCR-based tests for qualitative detection of HCV RNA.

1.      Amplicor Hepatitis C Virus Test

2.      Cobas Amplicor Hepatitis C Virus Test

These qualitative tests carry out in vitro nucleic acid amplification and detection of Hepatitis C Virus RNA genotypes 1 to 6 in human EDTA plasma or serum using the COBAS® AmpliPrep Instrument for automated specimen processing and the COBAS® TaqMan® Analyzer or the COBAS® TaqMan® 48 Analyzer for automated amplification and detection. Other commercially available non-approved assays are used by some diagnostic laboratories.

3.3. Quantitative Assays

The quantitative assays determine the quantity of HCV RNA in blood using signal amplification techniques (branched DNA assay). The level of HCV RNA in blood helps in predicting the likelihood of response to treatment, and the change in the level of HCV RNA during treatment can be used to monitor response. The results should be reported in international units to standardize data. Because a change in the HCV RNA level is used to monitor treatment response, it is important at the outset of treatment to obtain the actual level rather than simply a report indicating that the level exceeds an upper limit of detection, since HCV RNA levels sometimes are above the linear range of currently available assays. In addition, the same quantitative test should be used while on therapy to avoid confusion (Strader et al., 2004). The only quantitative test that has currently received FDA approval is Versant HCV RNA version 3.0 (Bayer Diagnostics, Tarrytown, NY) in given Table 3.1.

Table 3.1 Assays for quantitative test for HCV RNA in blood serum.

(Adapted from Strader et al., 2004)

3.4. HCV Genotyping

HCV has a high degree of genetic heterogeneity that has significantly complicated classification. Genotypes (equidistant phylogenetic groups) differ from each other by 31–33% of nucleotides. Whereas subtypes, the next level of classification, differ by 20–25% of nucleotides (Simmonds et al., 2005). All HCV genotypes share an identical complement of collinear genes of similar size in the large open reading frame. According to recent classification scheme seven distinct genotypes and 67 confirmed subtypes are described which play a critical role in determining the response to antiviral therapy (Smith et al., 2014).

Several methods are used to genotype HCV,  including direct nucleic acid sequencing, a reverse hybridization analysis using genotype-specific probes corresponding to 5' noncoding region sequences, subtype-specific reverse transcription (RT)-PCR, DNA restriction fragment length polymorphism (RFLP) analysis (Davidson et al,. 1995) of highly conserved 5' noncoding region, heteroduplex mobility analysis, primer-specific and mispair extension analysis, melting curve analysis with fluorescence resonance energy transfer probes, and serologic genotyping. Nucleic acid sequencing and phylogenetic analysis of an appropriate subgenomic region remains the reference method. All of these methods are technically complex and costly whereas many lack standardization. Therefore, there are two methods currently used for clinical purposes which are based on detection of sequence heterogeneity in the 5' noncoding (5'NC) region (Nolte et al., 2003).

1. TRUGENE HCV 5′NC Genotyping Kit

The Trugene assay may be used in a routine clinical laboratory, after amplification with the Amplicor assay, to genotype all the HCV isolates. The Trugene assay does not require an additional specimen-processing step and utilizes products obtained from a single, non-nested amplification reaction, thus eliminating delays and the risks of carryover contamination. It is based on direct sequencing followed by comparison with a reference sequence database. While sequence variability is found throughout the HCV genome, the 5′ noncoding (5′NC) region remains highly conserved and is considered the best target for detection by reverse transcription-PCR (RT-PCR) (Young et al., 1993; Smith et al., 1995).

All serum samples are stored at −70°C prior to testing. Samples are subjected to RNA extraction. Then qualitative Amplicor HCV test is performed in accordance with the manufacturer's instructions and amplifies a 244-nucleotide sequence within the 5′NC region of the HCV genome.  All specimens further analyzed by Trugene NS5B were processed with the Epicentre Masterpure RNA purification kit. These denatured amplicons from the Amplicor test were purified by using the QIAquick PCR purification kit. Next step involves amplification of a portion of the HCV NS5B region with the Titan One Tube RT-PCR kit and used directly in the assay. TRUGENE NS5B genotyping was performed with HCV NS5B RT-PCR amplification products directly in CLIP sequencing reactions. NS5B sequences were analyzed with the OpenGene DNA sequencing system, GeneObjects DNA analysis software, and a prototype HCV NS5B GeneLibrarian module 5.1. (Roque-Afonso et al., 2002).

2. Reverse Hybridization Line Probe Assay

It is based on reverse hybridization of PCR amplicons on a nitrocellulose strip coated with genotype-specific oligonucleotide probes. Once the genotype is identified, the test need not be repeated. Current commercial tests fail to identify the genotype in a small proportion such as <3 % of HCV-positive persons (Stuyver et al., 1993).

4.      Treatment of HCV

The basic purpose of treatment of HCV is to achieve a high rate of sustained virologic response (SVR). SVR is defined as an undetectable viral RNA after the completion of antiviral therapy (12 weeks after treatment completion). These days antiviral therapy is largely based on new direct-acting antiviral agents which includes the two newly FDA-approved drugs, sofosbuvir and simeprevir. Sofosbuvir is a uridine nucleotide analogue that selectively inhibits the HCV NS5B RNA-dependent RNA polymerase in HCV and advised in combination with several other drugs, with and without PEG-INF, against HCV infection. It targets the highly conserved nucleotide-binding pocket of this enzyme and functions as a chain terminator a nucleotide analog, with improved efficacy as well as a safety profile was formulated (Gane et al. 2013).

In patients with HCV genotype 2 or 3 infection for whom treatment with PEGinterferon and ribavirin was not an option, 12 or 16 weeks of treatment with sofosbuvir in combination with ribavirin proved highly effective. Efficacy was increased among patients with HCV genotype 2 infection and those without cirrhosis. In previously treated patients with genotype 3 infection, 16 weeks of therapy was significantly more effective than 12 weeks (Jacobson et al., 2013).  Sofosbuvir is also advised in combination with ribavirin and interferon to those patients who are infected with HCV genotype 1 or 4. It is also recommended in combination with ribavirin in HCV-infected patients with hepatocellular carcinoma who are awaiting liver transplantation (Rodriguez-Torres et al., 2013).

Sofosbuvir is found to be causing a significant changes in metabolism in recent studies such as a significant increase in low-density lipoprotein (LDL) and total cholesterol (TC) was observed after treatment (Morales et al., 2016). However, it offers many advantages due to its high potency, low side effects, oral administration, and high barrier to resistance. It is generally well-tolerated and may adversely cause headache, fatigue, nausea, dizziness, upper respiratory tract infections, rash, back pain and anemia, but exact safety profile can only be judged when this drug is actually used on a large scale.

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