Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries

Introduction

Hepatitis C virus (HCV) infection has emerged as one of the major health challenges with an estimated 200 million HCV-infected people covering about 3.3 per cent of the world population and is responsible for around 350,000 deaths per year12. HCV infection is a blood borne infection, which mainly causes liver fibrosis which advances into liver cirrhosis and liver cancer3. Low-resource regions such as India, East Asia, North Africa and the Middle East contribute to 80 per cent of the global burden of HCV infection4. Around nine per cent population of world's HCV-infected individuals is living in India and accounts for major share of HCV infected people (~12-18 million), the overall prevalence of HCV is considered low to moderate (1-1.5%) due to higher population size56. HCV is mainly transmitted through transfusion of infected blood and blood products. After transmission, the virus may remain in latent phase due to suppression by host immune system. However, during its infectious phase, the replication of HCV is very robust, and around 10 trillion virion particles can be produced per day7. HCV is an RNA virus and its genetic diversity is constantly evolving due to rapid globalization, which ultimately results in varying therapeutic response in different geographical regions. On the basis of genomic variability, HCV is classified into seven major genotypes and 67 subtypes8. In India, HCV is responsible for acute viral hepatitis in up to 21 per cent910 and chronic liver disease in 14-26 per cent cases of HCV infections1011. Epidemiological studies have shown that the persistence of HCV infection is a major risk for development of hepatocellular carcinoma (HCC)12. HCC is the fifth most common cancer and a major cause of death in patients with chronic HCV infection and responsible for approximately one million deaths each year1314. Earlier, the available antiviral therapies were effective in the treatment of HCV infection only at early stages and its identification and treatment at chronic stage were generally meaningless. But now, with the advancement in medical research, newer anti-HCV drugs have been developed which are also useful in the advanced cirrhosis caused by HCV1516.

HCV can be detected by serological tests employing anti-HCV antibodies and molecular tests using molecular probes complementary to HCV RNA sequence17. As per Drugs and Cosmetics Act, 1940 and 1945 and rules thereunder, the screening of blood units with third-generation serological assays is mandatory in India18. Fourth-generation serological assays with higher sensitivities are available which claim to reduce the window period. The molecular assays not only help in the early diagnosis but also confirm the infection stage. The diagnosis and quantification of HCV with highly sensitive and specific method play a crucial role in the management of HCV infection. It has been reported that different genotypes of HCV respond differently to anti-HCV therapy19. These genotypes differ from each other at their genomic levels. HCV is prone to transcriptional variations during the process of replication of its genome. In addition, the genome of HCV is constantly evolving due to globalization. This review summarizes information on HCV molecular diagnostic methods and their applications used in various parts of the world.

HCV genome organization and function

HCV is a member of family Flaviviridae and has about 9.6 kb positive-sense single-stranded RNA genome. The HCV genome contains single open reading frame (ORF) flanked by highly structured untranslated regions (UTRs) which are essential for RNA replication (Fig. 1)20. This single ORF codes for three structural and six non-structural proteins. The structural proteins are core protein (C) and two envelope proteins E1 and E2. Non-structural proteins are named as NS2, NS3, NS4A, NS4B, NS5A and NS5B. Two proteins p7 and F are also encoded due to frame shifting in the coding region. Core protein of HCV is an RNA binding basic protein, which forms viral capsid. It is released as a 191 amino acid (aa) precursor of around 23-kDa molecular weight containing three conserved domains (122 aa N terminal hydrophilic domain, 50 aa hydrophobic C terminal domain and 20 aa signal peptide domain)21. Besides viral capsid formation, it also interacts with various cellular proteins and pathways that play an important role in HCV life cycle22. E1, E2 and type-1 transmembrane proteins are crucial components of viral envelope and are also essential for the viral entry and fusion with host cell2324. During early phases of viral infection, E2 plays a vital role by initiating interaction with one or several components of the receptor complex2526. Function of E1 is not clearly understood, but it is hypothesized to be involved in the intracytoplasmic viral-membrane fusion2526.

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Fig. 1

Schematic representation of genome organization of hepatitis C virus. The entire genome encodes a polyprotein, which is further processed into three structural [core (C), envelop protein (E1 & E2)] and five non-structural proteins [NS1, NS2, NS3, NS4 & NS5]; Protein positions are shown by numbers on the upper part of the scheme. Figure modified and reproduced with permission from Ref 20.

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Non-structural protein, NS2 is a 21-23 kDa transmembrane protein that contains two internal sequences, which are responsible for ER membrane association27. NS2 is a short-lived protein and acts as a zinc-dependent metalloprotease with NS3 amino-terminal domain202829. NS3 is a multifunctional protein having serine protease domain and a helicase/NTPase domain in its N and C terminal, respectively. The 21-30 aa central region of NS4A acts as a cofactor for protease activity of NS3. This complementary interaction of NS3-NS4A is attractive target for the anti-HCV therapy. The anti-HCV therapies are designed to disrupt this interaction to break infectious pathway1930. NS4B is a 261 aa membrane protein having four transmembrane domains and helps in membrane association for replication complex3132, inhibits cellular synthesis3334 and modulates RNA-dependent RNA polymerase (RdRp) activity of NS5B35. NS5A, a 56-58 kDa phosphorylated zinc-metalloprotein, plays an important role in virus replication and regulation of cellular pathways. NS5A provides the resistance against interferon by binding to interferon-α, protein kinase R (PKR) and inhibits its antiviral effect36. NS5B, an RdRp, is a tail-anchored protein3738 and forms classical ‘fingers, palm and thumb’ structure. The interaction between the fingers and thumb subdomains is essential for the complete catalytic cycle to permit synthesis of positive and negative RNA strand of HCV39. The p7, a 63 aa small polypeptide, contains two transmembrane α-helix domains connected by a cytoplasmic loop. A mutation or deletion in its cytoplasmic loop suppresses HCV infectivity in intraliver transfections40.

Natural history of hepatitis C virus

The understanding of the viral persistence and modes of transmission is important for the prevention of HCV infection. HCV is generally transmitted through transfusion of infected blood, unprotected high-risk sexual activity, organ transplantation from infected donor and mother to foetus. With the implementation of improved screening methods, reduction in HCV transmission from 0.0002 to 0.00005 per cent has been observed in developed countries414243.

The better understanding of the viral natural history plays an important role in the viral management and disease outcome. In a population, infected with HCV, it has been observed that in 15-45 per cent individuals, the infection gets spontaneously cleared within six months while in 55-85 per cent of the individuals, it persists. In acute phase, 70-80 per cent of the HCV-infected individuals are asymptomatic and unaware about the infection till it reaches the chronic phase. Of the infected individuals, 75-85 per cent may develop chronic infection which advances into liver fibrosis, cirrhosis, extrahepatic complications and HCC44. The characteristics of persistence of HCV infection and development of the chronic infection may differ from individual to individual depending on factors such as age, immune response, degree of inflammation, HIV/HBV coinfection or lifestyle habit such as consumption of alcohol44. According to a study performed by Kiyosawa et al45, the average time taken for the development of chronic hepatitis was about 10 yr, development of cirrhosis was 21 yr and development of HCC was around 29 years. With an estimate around 10-15 per cent of HCV-infected cases will progress into liver cirrhosis within first 20 yr, which increases the risk of developing HCC44.

HCV genotypes, subtypes and anti-HCV therapy

As mentioned previously, HCV strains are classified into seven genotypes based on the phylogenetic and sequence analyses of HCV genomes, and within each genotype, HCV is further classified into 67 subtypes46. In India, genotype 3 is the predominant genotype (~63.85%) followed by genotype 1 (25.72%). HCV genotype 3 is more prevalent in the northern, eastern and western parts of India and genotype 1 is more prevalent in the southern India47. Despite, the widespread prevalence of genotypes 3 and 1, there also exists increased prevalence of genotypes 4 and 6 in certain regions of the country. Genotype 4 is found mostly in south Indian patients from the States of Andhra Pradesh and Tamil Nadu. Genotype 6 is found to be prevalent in patients belonging to northeastern parts of India48. An increased prevalence of genotype 6 in various parts of the neighbouring country of Myanmar which shares its boundaries with the northeastern States of India, has also been observed49. Genotype 2 is rarely reported while genotype 5 is yet to be reported from India47.

With the advancement in medical research and clinical trials of new drugs, the treatment of HCV infection has been shifted from pegylated interferon/ribavirin to direct-acting antiviral (DAA) combination therapies. These DAAs target and inhibit key stages of the HCV replication pathway. DDAs are classified into four classes on the basis of their mechanism of action and target50 (Table).

Table Classes of direct-acting antivirals (DDA) approved/under clinical trials

The combined drugs therapy may involve the use of two to three drugs515253. In 2011, DAAs, telaprevir and boceprevir were approved by the US Food and Drug Administration (FDA) for anti-HCV therapy54. These first-generation DAAs inhibit NS3/4A protease during their action against HCV. However, both were withdrawn from US market after three years in 2014. HCV infection treatment with protease inhibitors (PIs) has shown better results with significant side effects55. However, during clinical trials, second-generation PIs have shown better results with the fewer side effects in comparison to first-generation DAAs. In 2016, two new combinations of DAAs, sofosbuvir/velpatasvir and grazoprevir/elbasvir were approved licensed in Europe and USA56. At present, three regimens are recommended for the treatment of patients infected with HCV genotype 1. These regimens are simeprevir plus sofosbuvir with or without ribavirin, ledipasvir/sofosbuvir and ombitasvir/paritaprevir/ritonavir plus dasabuvir with or without ribavirin57. Ghany et al58 reported 35-70 per cent increased response with the use of triple combination therapy in comparison to double combination therapy for treatment of HCV genotype 1. In case of genotypes 2 and 3, regimen of sofosbuvir and ribavirin is recommended. For HCV genotypes 4, 5 and 6 and HIV co-infection, ledipasvir/sofosbuvir and ombitasvir/paritaprevir/ritonavir plus dasabuvir with or without ribavirin have been recommended by both the American Association for the Study of Liver Diseases and the Infectious Diseases Society of America575960. Both of these regimens have also been approved by US-FDA for HCV-infected patients coinfected with HIV.

HCV genotyping methods

The genotype of HCV for diagnosis is mostly determined by sequencing of genomic nucleotide sequence or by kit-based assays which employ complementary probes to report genotype present in a specimen. Sequencing of highly conserved regions such as NS5, core, E1 and 5’UTR is the most commended method used for genotyping of HCV61. The kit-based assays are easy-to-use and do not require expertize as is required for sequencing. Both TruGene 5’NC HCV Genotyping kit (Siemens Healthcare Diagnostics Division, Tarrytown, NY) and Versant™ HCV Genotype Assay LiPA (version I, Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany) are based on 5’UTR sequence. Both these kits use reverse hybridization with genotype-specific oligonucleotide probes62. Both kits claim to detect HCV genotypes 1 to 6. Abbott RealTime HCV Genotype II Kit claims to detect genotype 1 to 5. HCV genotype kit cobas® HCV GT, manufactured by Roche claims to identify HCV genotypes 1-6 and can discriminate between subtype a and b of genotype-1. Fully automated system cobas® 4800 (Roche, USA) has also been launched for HCV genotyping. The complementary probes against three regions, 5’UTR, Core and NS5B of HCV genome have been incorporated for genotyping and subtyping accuracy.

Duarte et al63 developed xMAP Luminex assay system, a liquid microarray-based HCV genotyping method. The complementary sequences against most variable region NS5B and highly conserved 5’UTR were used to determine HCV genotypes. The results shown were in 100 per cent concordance with Versant™ HCV Genotype Assay LiPA. Two high-throughput methods like LightCycler® systems based on metlting curve analysis6465 and using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) technology have also been developed66. Athar et al67 developed a real-time polymerase chain reaction (PCR)-based assay using two dual-labelled self-quenched probes of core region of HCV genome. They used the specific probes of the HCV core region and exact HCV genotypes could be determined by melting curves from the respective probes in RT PCR67.

Other alternative HCV genotyping methods are amplification with type-specific primers or type-specific probes6869, restriction fragment length polymorphism (RFLP) analysis70, line-probe testing71, and heteroduplex mobility analysis7273.

Diagnostic methods for HCV

HCV infection can be detected using serological methods and molecular methods (Fig. 2)74. Accurate diagnosis of HCV infection is important before initiating therapy. The diagnosis to know the stage of HCV infection is also crucial because HCV infection gets cleared within 6-12 months in 15-45 per cent of infected individuals and these individuals remain anti-HCV antibody positive without any detectable viraemia. In such cases, physicians must differentiate between the infected and recovered individuals75. Earlier, the serological reactivity of HCV infection by ELISA has been confirmed by recombinant immunoblot assay (RIBA). RIBA has been discontinued as a confirmatory method for HCV infection diagnosis. Nowadays, nucleic acid testing (NAT) is considered as the gold standard to confirm active HCV infection76. NAT is based on the detection of HCV RNA or HCV genome in a specimen.

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Fig. 2

Algorithm for hepatitis C virus testing. Detection of HCV antibody (Ab) is done by laboratory assay. Further testing of HCV Ab positive samples will show either current or past HCV infection or false HCV antibody positivity. Positive samples should be tested by nucleic acid-based testing (NAT), as positive indicates current HCV infection and negative indicates either past or resolved HCV infection, or false Ab positivity. Modified and reproduced from Ref 71.

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Conclusion

The nucleic acid-based testing for HCV has reduced its window period to around one week. This technique has proved its advancement and needs over conventional serological methods99. Therefore, the implementation of NAT will be of great benefit to low-resource countries due to high prevalence of HCV.