|Year : 2018 | Volume
| Issue : 5 | Page : 445-455
Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries
Anoop Kumar, Manoj Kumar Rajput, Deepika Paliwal, Aakanksha Yadav, Reba Chhabra, Surinder Singh
National Institute of Biologicals, Noida, India
|Date of Submission||15-Nov-2016|
|Date of Web Publication||1-Aug-2018|
Dr Anoop Kumar
Molecular Diagnostic Laboratory, National Institute of Biologicals, A-32, Sector-62, Noida 201 309, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Hepatitis C virus (HCV) infection is a blood borne and transfusion-transmitted infection (TTI). It has emerged as one of the major health challenges worldwide. In India, around 12-18 million peoples are infected with HCV, but in terms of prevalence percentage, its looks moderate due to large population. The burden of the HCV infection increases due to lack of foolproof screening of blood and blood products before transfusion. The qualified screening and quantification of HCV play an important role in diagnosis and treatment of HCV-related diseases. If identified early, HCV infection can be managed and treated by recently available antiviral therapies with fewer side effects. However, its identification at chronic phase makes its treatment very challenging and sometimes ineffective. The drugs therapy for HCV infection treatment is also dependent on its genotype. Different genotypes of HCV differ from each other at genomic level. The RNA viruses (such as HCV) are evolving perpetually due to interaction and integration among people from different regions and countries which lead to varying therapeutic response in HCV-infected patients in different geographical regions. Therefore, proper diagnosis for infecting virus and then exact determination of genotype become important for targeted treatment. This review summarizes the general information on HCV, and methods used for its diagnosis and genotyping.
Keywords: Hepatitis - hepatitis C virus - hepatitis C virus genotypes - molecular diagnostic - nucleic acid testing - serological assay
|How to cite this article:|
Kumar A, Rajput MK, Paliwal D, Yadav A, Chhabra R, Singh S. Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries. Indian J Med Res 2018;147:445-55
|How to cite this URL:|
Kumar A, Rajput MK, Paliwal D, Yadav A, Chhabra R, Singh S. Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource countries. Indian J Med Res [serial online] 2018 [cited 2019 Jul 23];147:445-55. Available from: http://www.ijmr.org.in/text.asp?2018/147/5/445/238238
| 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 year,. HCV infection is a blood borne infection, which mainly causes liver fibrosis which advances into liver cirrhosis and liver cancer. 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 infection. 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 size,. 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 day. 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 subtypes. In India, HCV is responsible for acute viral hepatitis in up to 21 per cent, and chronic liver disease in 14-26 per cent cases of HCV infections,. Epidemiological studies have shown that the persistence of HCV infection is a major risk for development of hepatocellular carcinoma (HCC). 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 year,. 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 HCV,.
HCV can be detected by serological tests employing anti-HCV antibodies and molecular tests using molecular probes complementary to HCV RNA sequence. 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 India. 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 therapy. 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 [Figure 1]. 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). Besides viral capsid formation, it also interacts with various cellular proteins and pathways that play an important role in HCV life cycle. 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 cell,. During early phases of viral infection, E2 plays a vital role by initiating interaction with one or several components of the receptor complex,. Function of E1 is not clearly understood, but it is hypothesized to be involved in the intracytoplasmic viral-membrane fusion,.
|Figure 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.|
Click here to view
Non-structural protein, NS2 is a 21-23 kDa transmembrane protein that contains two internal sequences, which are responsible for ER membrane association. NS2 is a short-lived protein and acts as a zinc-dependent metalloprotease with NS3 amino-terminal domain,,. 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 pathway,. NS4B is a 261 aa membrane protein having four transmembrane domains and helps in membrane association for replication complex,, inhibits cellular synthesis, and modulates RNA-dependent RNA polymerase (RdRp) activity of NS5B. 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 effect. NS5B, an RdRp, is a tail-anchored protein, 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 HCV. 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 transfections.
| 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 countries,,.
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 HCC. 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 alcohol. According to a study performed by Kiyosawa et al, 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 HCC.
| 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 subtypes. 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 India. 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 India. 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 observed. Genotype 2 is rarely reported while genotype 5 is yet to be reported from India.
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 target [Table 1].
|Table 1: Classes of direct-acting antivirals. (DDA) approved/under clinical trials|
Click here to view
The combined drugs therapy may involve the use of two to three drugs,,. In 2011, DAAs, telaprevir and boceprevir were approved by the US Food and Drug Administration (FDA) for anti-HCV therapy. 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 effects. 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 USA. 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 ribavirin. Ghany et al 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 America,,. 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 HCV. 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 probes. 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 al 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 analysis, and using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) technology have also been developed. Athar et al 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 PCR.
Other alternative HCV genotyping methods are amplification with type-specific primers or type-specific probes,, restriction fragment length polymorphism (RFLP) analysis, line-probe testing, and heteroduplex mobility analysis,.
| Diagnostic Methods for Hcv|| |
HCV infection can be detected using serological methods and molecular methods [Figure 2]. 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 individuals. 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 infection. NAT is based on the detection of HCV RNA or HCV genome in a specimen.
|Figure 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. |
Click here to view
Serological assays for HCV diagnosis
HCV can be diagnosed by the detection of the anti-HCV antibody in the serum or plasma samples, where a non-reactive result is considered as absence of HCV infection. Further, to declare an HCV infection as active infection, it needs to be confirmed by HCV RNA test. Assays used for the detection of the HCV infection have been evolved to minimize the number of false positives with improvement in their specificity. Successive generations of the HCV serological assays have not only improved sensitivity and specificity but have also reduced the window period for the detection of infection,,.
- First-generation assay: These were based on binding of anti-HCV antibody and recombinant protein-containing NS4 (C100-3) epitopic region. These assays could identify about 80 per cent anti-HCV IgG in post-transfusion cases; therefore, their sensitivity and specificity were very low. Their false positivity rate in low-risk populations has been reported to be as high as 60 per cent.
- Second-generation assays: The recombinant proteins from core (structural), NS3 and NS4 were used as binding proteins with anti-HCV antibody. These assays have been reported to have better sensitivity and specificity. Second-generation assays reduced window period of HCV to 10-24 wk.
- Third-generation assays: The immunosorbent contains recombinant proteins of core, NS3, NS4 and NS5 regions. The diagnosis specificity of more than 99 per cent has been reported for this category of assays. Third-generation assays can show false-negative results in the patients who are immunocompromised or undergoing haemodialysis. In India, as per Drugs and Cosmetics act, 1940 and 1945, the use of third-generation serological assays is mandatory for screening of blood donor units for HCV infection before transfusion.
- Fourth-generation assays: Fourth-generation assays are also known as ‘antigen-antibody combo’ tests because the principle of these assays is based on the detection of both anti-HCV antibody and HCV antigen at the same time. These assays are highly sensitive and are responsible for high reduction in window period. The average window period is 26.8 days for these assays.
Earlier, according to CDC, the person having positive serological test had to be confirmed by RIBA for HCV infection, but now HCV RNA nucleic acid testing is used to confirm active HCV infection.
Recombinant immunoblot assays (RIBA)
RIBA has previously been used for confirmation of the HCV infection and it was based on the detection of anti-HCV antibody by immobilized HCV antigen and synthetic peptide from structural (core) and non-structural (NS3 and NS5) proteins as individual bands onto a membrane. The appearance of at least two bands was considered as indicator for confirmed infection and appearance of single band was considered as indeterminate result. Indeterminate cases were due to non-specific cross-reacting antibodies or the broad humoral response had not be triggered by the recent infection. In general, the results of one-week old HCV-infected individuals were reported as indeterminate who might be converted into confirmed cases after 1-6 months of infection.
Rapid tests are configured to generate results within half an hour and can be used for point-of-care testing. These assays do not require sophisticated instrument and highly trained staff. Three rapid tests (Orasure, Chembio and Medmira) for anti-HCV IgG have been evaluated by the CDC in laboratory as well as in field settings. The specificities and sensitivities of these tests were >99 per cent and 86-99 per cent, respectively. The main disadvantage in serological assays is their inability to discriminate between active and non-active infection. Serological assays test positive due to the presence of anti-HCV IgG even after the clearance of viraemic particles by immune system. The other drawback of these assays is large window period. During this period, HCV RNA is the first detectable diagnostic marker and is followed by core antigen diagnostic marker. The generation of anti-HCV antibody is followed by these two diagnostic markers. Due to this drawback, blood bank in the USA adopted nucleic acid-based testing (NAT) as a gold standard method for HCV diagnosis in late 1990s and due to implementation of NAT, the transfusion-related risk of HCV infection has been reduced to 0.0001 per cent in USA.
Nucleic acid testing (NAT)
NAT is a molecular technique and is based on amplification and detection of viral nucleic acid. NAT reduces the risk of transfusion transmitted infections (TTIs); therefore, it adds an additional layer of safety to the blood to be used for transfusion. Two types of technologies, PCR and transcription-mediated amplification (TMA) are routinely used for NAT testing of blood donations for HCV infection. The main advantage of the nucleic acid testing is the early detection of HCV infection. HCV RNA in infected samples can be detected within one week of initial exposure. Due to high sensitivity and specificity, NAT is used for the resolution of samples which are reported as indeterminate and or false negative by serological methods.
In an international survey on NAT testing of about 300 million blood donations for HIV and HCV and about 100 million donations for HBV infection in 37 countries, Roth et al reported 3000 NAT yield. These blood donations were reported as non-reactive by serological screening methods.
NAT blood screening experience in low-resource countries
Realizing the need and benefits of NAT testing, low-resource countries are also implementing NAT testing on national or centre level for screening blood donation. In 2007, at Thai blood centre, 486,676 seronegative blood donations were screened by NAT. The NAT yield rates for HIV-1, HCV and HBV were 1:97,000, 1:490,000 and 1:2800, respectively. In north India at a blood bank, 73,898 blood donations were screened for HIV, HBV and HCV by ELISA and individual donor-nucleic acid amplification test (ID-NAT). Out of all donations, 1104 (1.49%) donations were found positive by multiplex NAT. Furthermore, 37 HCV, 73 HBV, one HIV and 10 HBV-HCV co-infected cases were reported by NAT testing who were non-reactive by ELISA testing. In India, different groups at different centres reported different NAT yield rates for HCV infection. The NAT yield reported by Agarwal et al was 1 in 610. Kumar et al reported 1 in 753 and Mangwana and Bedi as 1 in 974. In Brazil till July 2005, 139,678 donations were screened in pools of 6 to 12 donations by in-house RT-PCR method having sensitivity of 500 IU/ml for all six genotypes of HCV. Out of 139,678 donations, 315 (0.23%) were found to be positive for HCV RNA.
The NAT testing can be performed for individual donations (ID-NAT) or min-pools (MP-NAT). A mini-pool may be prepared by pooling 6, 8, 16 or 24 individual donations. Although NAT is very beneficial due to the requirement of higher investment for its implementation, low-resources countries like India are not able to make it mandatory for screening of blood for TTIs. To make blood screening cost-effective, different counties and institutions are using mini-pools of 6, 8, 16 and 24. In Germany, after introduction of MP-NAT in 1997, 23 HCV, 2 HIV-1 and 43 HBV-infected blood donation were detected in around 31 million blood donations. Based on the data analysis of NAT, Mathur et al have shown that NAT can be made cost-effective by pooling method (mini-pools) in India. There are some disadvantages of mini-pools in NAT testing; first, dilution of the viral copy number decreases sensitivity. Second, if a pool is found reactive, all pooled donations need to be tested for the identification of the individual positive donation. Therefore, to overcome these limitations, ID-NAT testing has been suggested.
Testing of HCV infection by NAT for its RNA detection is done by PCR or TMA or branched deoxyribonucleic acid (bDNA) signal amplification. In NAT testing technology of HCV, highly conserved 5’ UTR of HCV genome is used as a target sequence for its detection. This region is conserved among all genotypes of HCV; therefore, primers are designed to hybridize with the least possible discrepancies among six different HCV genomes. Most of the manufacturers of NAT testing kits have developed their assays compatible only to their equipment. All types of platforms, manual, semi-automated and fully automated are available for HCV NAT testing. In research laboratories, qualitative HCV-NAT is used for diagnosis using conventional RT-PCR, but diagnostics laboratories have started using kits for diagnosis of HCV.
The qualitative NAT has been used for screening of HCV infection during blood donation and to confirm viraemia in patients. The lower limit of detection of ≤50 IU/ml is claimed by various manufacturers for their qualitative molecular methods. On the other hand, viral load determination of HCV disease prognosis can be accomplished by quantitative RT-PCR and bDNA technology.
The prevalence of HCV in India has been higher in comparison to Western Europe and USA. The HCV prevalence reported by various groups in Indian population is 1 in 4138, 1 in 1997 and 1 in 2536.
Although NAT reduces the risk of TTIs such as HCV in the Indian context, there are some limitations for its mandatory implementation. In India, the use of NAT in regular practises has been restricted due to high cost, technical demand such as trained workforce or equipment consumables. In 2014, Chandrashekar developed an in-house assay for safety of blood at a minimal cost. This assay was found feasible to implement in a small molecular biology unit in any types of blood banks.
The introduction of the NAT to detect window period donations in 300,000 to 3,000,000 blood donations, could save around three lives in the developed countries, whereas in developing countries like India it may be 300 to 3000 times more beneficial as compared to developed countries.
| 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 methods. Therefore, the implementation of NAT will be of great benefit to low-resource countries due to high prevalence of HCV.
Financial support & sponsorship: None.
Conflicts of Interest: None.
| References|| |
Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect
Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: New estimates of age-specific antibody to HCV seroprevalence. Hepatology
Farag MM, Sofy AR, Mousa AA, Ahmed MA, Alganzory MR. Molecular assay and genotyping of hepatitis C virus among infected Egyptian and Saudi Arabian patients. Virology
) 2015; 6
Jayasekera CR, Barry M, Roberts LR, Nguyen MH. Treating hepatitis C in lower-income countries. N Engl J Med
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin
Selvarajah S, Busch MP. Transfusion transmission of HCV, a long but successful road map to safety. Antivir Ther
Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, et al.
Hepatitis C viral dynamics in vivo
and the antiviral efficacy of interferon-alpha therapy. Science
Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, et al.
Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource. Hepatology
Arankalle VA, Chadha MS, Jha J, Amrapurkar DN, Banerjee K. Prevalence of anti-HCV antibodies in Western India. Indian J Med Res
Kar P. World Hepatitis Day-new challenges. Indian J Med Res
Mehta SK, Singh V, Bhasin DK, Kumar YR, Kochhar R. Hepatitis C virus in patients with acute and chronic liver disease. Indian J Gastroenterol
El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology
European Association For The Study Of The Liver; European Organisation For Research And Treatment Of Cancer. EASL-EORTC clinical practice guidelines: Management of hepatocellular carcinoma. J Hepatol
Mitra AK. Hepatitis C-related hepatocellular carcinoma: Prevalence around the world, factors interacting, and role of genotypes. Epidemiol Rev
Morishima C, Gretch DR. Clinical use of hepatitis C virus tests for diagnosis and monitoring during therapy. Clin Liver Dis
Richter SS. Laboratory assays for diagnosis and management of hepatitis C virus infection. J Clin Microbiol
Clemens JM, Taskar S, Chau K, Vallari D, Shih JW, Alter HJ, et al.
IgM antibody response in acute hepatitis C viral infection. Blood
Pawlotsky JM. New therapeutic strategies in chronic hepatitis C. Gastroenterol Clin Biol
Echeverría N, Moratorio G, Cristina J, Moreno P. Hepatitis C virus genetic variability and evolution. World J Hepatol
Grakoui A, Wychowski C, Lin C, Feinstone SM, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol
McLauchlan J. Properties of the hepatitis C virus core protein: A structural protein that modulates cellular processes. J Viral Hepat
Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med
Nielsen SU, Bassendine MF, Burt AD, Bevitt DJ, Toms GL. Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver. J Gen Virol
Flint M, McKeating JA. The role of the hepatitis C virus glycoproteins in infection. Rev Med Virol
Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, et al.
A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci U S A
Yamaga AK, Ou JH. Membrane topology of the hepatitis C virus NS2 protein. J Biol Chem
Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice CM. Characterization of the hepatitis C virus-encoded serine proteinase: Determination of proteinase-dependent polyprotein cleavage sites. J Virol
Hijikata M, Mizushima H, Tanji Y, Komoda Y, Hirowatari Y, Akagi T, et al.
Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A
Bartenschlager R, Lohmann V, Wilkinson T, Koch JO. Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. J Virol
Elazar M, Liu P, Rice CM, Glenn JS. An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication. J Virol
Gretton SN, Taylor AI, McLauchlan J. Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci. J Gen Virol
Florese RH, Nagano-Fujii M, Iwanaga Y, Hidajat R, Hotta H. Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus. Virus Res
Kato J, Kato N, Yoshida H, Ono-Nita SK, Shiratori Y, Omata M, et al.
Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo
. J Med Virol
Piccininni S, Varaklioti A, Nardelli M, Dave B, Raney KD, McCarthy JE, et al.
Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J Biol Chem
Gale M Jr., Katze MG. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol Ther
Ivashkina N, Wölk B, Lohmann V, Bartenschlager R, Blum HE, Penin F, et al.
The hepatitis C virus RNA-dependent RNA polymerase membrane insertion sequence is a transmembrane segment. J Virol
Schmidt-Mende J, Bieck E, Hugle T, Penin F, Rice CM, Blum HE, et al.
Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem
Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC, et al.
Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol
Sakai A, Claire MS, Faulk K, Govindarajan S, Emerson SU, Purcell RH, et al.
The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences. Proc Natl Acad Sci U S A
Alter HJ, Liang TJ. Hepatitis C: The end of the beginning and possibly the beginning of the end. Ann Intern Med
Dodd RY, Notari EP 4th
, Stramer SL. Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population. Transfusion
Roth WK, Weber M, Buhr S, Drosten C, Weichert W, Sireis W, et al.
Yield of HCV and HIV-1 NAT after screening of 3.6 million blood donations in central Europe. Transfusion
Chen SL, Morgan TR. The natural history of hepatitis C virus (HCV) infection. Int J Med Sci
Kiyosawa K, Tanaka E, Sodeyama T, Furuta K, Usuda S, Yousuf M, et al.
Transition of antibody to hepatitis C virus from chronic hepatitis to hepatocellular carcinoma. Jpn J Cancer Res
Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, et al.
Global distribution and prevalence of hepatitis C virus genotypes. Hepatology
Bhattacharya PK, Roy A. Management of hepatitis C in Indian context: An update. J Liver
Christdas J, Sivakumar J, David J, Daniel HD, Raghuraman S, Abraham P, et al.
Genotypes of hepatitis C virus in the Indian sub-continent: A decade-long experience from a tertiary care hospital in South India. Indian J Med Microbiol
Wasitthankasem R, Vongpunsawad S, Siripon N, Suya C, Chulothok P, Chaiear K, et al
. Genotypic distribution of hepatitis C virus in Thailand and Southeast Asia. PLoS One
Zopf S, Kremer AE, Neurath MF, Siebler J. Advances in hepatitis C therapy: What is the current state - What come's next? World J Hepatol
Feld JJ, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature
Jun DW, Tak WY, Bae SH, Lee YJ. Recent trends in the treatment of chronic hepatitis C. Korean J Hepatol
McHutchison JG, Lawitz EJ, Shiffman ML, Muir AJ, Galler GW, McCone J, et al
. Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection. N Engl J Med
Geddawy A, Ibrahim YF, Elbahie NM, Ibrahim MA. Direct acting anti-hepatitis C virus drugs: clinical pharmacology and future direction. J Transl Int Med
US Food and Drug Administration. Approval of Incivek (telaprevir) a Direct Acting Antiviral drug (DAA) to Treat Hepatitis C Virus (HCV)
. Available from: http://www.natap.org/2011/HCV/telap_02.htm
, accessed on April 30, 2018.
Pawlotsky JM. Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens. Gastroenterology
American Association for the Study of Liver Diseases and the Infectious Diseases Society of America. Recommendations for Testing Managing, and Treating Hepatitis C
. Available from: http://www.hcvguidelines.org
, accessed on April 20, 2017.
Ghany MG, Nelson DR, Strader DB, Thomas DL, Seeff LB; American Association for Study of Liver Diseases. An update on treatment of genotype 1 chronic hepatitis C vi
rus infection: 2011 practice guideline by the
American Association for the Study of Liver Diseases. Hepatology
Osinusi A, Townsend K, Kohli A, Nelson A, Seamon C, Meissner EG, et al.
Virologic response following combined ledipasvir and sofosbuvir administration in patients with HCV genotype 1 and HIV co-infection. JAMA
Sulkowski MS, Vargas HE, Di Bisceglie AM, Kuo A, Reddy KR, Lim JK, et al.
Effectiveness of simeprevir plus sofosbuvir, with or without ribavirin, in real-world patients with HCV genotype 1 infection. Gastroenterology
Ohno T, Lau JY. The “gold-standard,” accuracy, and the current concepts: Hepatitis C virus genotype and viremia. Hepatology
Ghany MG, Strader DB, Thomas DL, Seeff LB; American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C: An update. Hepatology
Duarte CA, Foti L, Nakatani SM, Riediger IN, Poersch CO, Pavoni DP, et al.
A novel hepatitis C virus genotyping method based on liquid microarray. PLoS One
. pii: e12822.
Haverstick DM, Bullock GC, Bruns DE. Genotyping of hepatitis C virus by melting curve analysis: Analytical characteristics and performance. Clin Chem
Schröter M, Zöllner B, Schäfer P, Landt O, Lass U, Laufs R, et al.
Genotyping of hepatitis C virus types 1, 2, 3, and 4 by a one-step LightCycler method using three different pairs of hybridization probes. J Clin Microbiol
Ilina EN, Malakhova MV, Generozov EV, Nikolaev EN, Govorun VM. Matrix-assisted laser desorption ionization-time of flight (mass spectrometry) for hepatitis C virus genotyping. J Clin Microbiol
Athar MA, Xu Y, Xie X, Xu Z, Ahmad V, Hayder Z, et al.
Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay. J Virol Methods
Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, et al.
Typing hepatitis C virus by polymerase chain reaction with type-specific primers: Application to clinical surveys and tracing infectious sources. J Gen Virol
(Pt 3) : 673-9.
Qu D, Li JS, Vitvitski L, Mechai S, Berby F, Tong SP, et al.
Hepatitis C virus genotypes in France: Comparison of clinical features of patients infected with HCV type I and type II. J Hepatol
Park YS, Lee KO, Oh MJ, Chai YG. Distribution of genotypes in the 5’ untranslated region of hepatitis C virus in Korea. J Med Microbiol
Stuyver L, Wyseur A, van Arnhem W, Hernandez F, Maertens G. Second-generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol
Margraf RL, Erali M, Liew M, Wittwer CT. Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis. J Clin Microbiol
White PA, Zhai X, Carter I, Zhao Y, Rawlinson WD. Simplified hepatitis C virus genotyping by heteroduplex mobility analysis. J Clin Microbiol
Centers for Disease Control and Prevention (CDC). Testing for HCV infection: An update of guidance for clinicians and laboratorians. MMWR Morb Mortal Wkly Rep
Micallef JM, Kaldor JM, Dore GJ. Spontaneous viral clearance following acute hepatitis C infection: A systematic review of longitudinal studies. J Viral Hepat
Dow BC, Buchanan I, Munro H, Follett EA, Davidson F, Prescott LE, et al
. Relevance of RIBA-3 supplementary test to HCV PCR positivity and genotypes for HCV confirmation of blood donors. J Med Virol
Barrera JM, Francis B, Ercilla G, Nelles M, Achord D, Darner J, et al.
Improved detection of anti-HCV in post-transfusion hepatitis by a third-generation ELISA. Vox Sang
Busch MP, Korelitz JJ, Kleinman SH, Lee SR, AuBuchon JP, Schreiber GB, et al.
Declining value of alanine aminotransferase in screening of blood donors to prevent posttransfusion hepatitis B and C virus infection. The Retrovirus Epidemiology Donor Study. Transfusion
Marwaha N, Sachdev S. Current testing strategies for hepatitis C virus infection in blood donors and the way forward. World J Gastroenterol
Barrera JM, Bruguera M, Ercilla MG, Sánchez-Tapias JM, Gil MP, Costa J, et al.
Incidence of non-A, non-B hepatitis after screening blood donors for antibodies to hepatitis C virus and surrogate markers. Ann Intern Med
Colin C, Lanoir D, Touzet S, Meyaud-Kraemer L, Bailly F, Trepo C, et al.
Sensitivity and specificity of third-generation hepatitis C virus antibody detection assays: An analysis of the literature. J Viral Hepat
Gerlach JT, Diepolder HM, Zachoval R, Gruener NH, Jung MC, Ulsenheimer A, et al.
Acute hepatitis C: High rate of both spontaneous and treatment-induced viral clearance. Gastroenterology
Smith BD, Drobeniuc J, Jewett A, Branson BM, Garfein RS, Teshale E, et al.
Evaluation of three rapid screening assays for detection of antibodies to hepatitis C virus. J Infect Dis
Donahue JG, Munoz A, Ness PM, Brown DE Jr, Yawn DH, McAllister HA Jr, et al
. The declining risk of post-transfusion hepatitis C virus infection. N Engl J Med
Yaseen SG, Ahmed SA, Johan MF, Kiron R, Daher AM. Evaluation of serological transfusion-transmitted viral diseases and mutliplex nucleic acid testing in Malaysian blood donors. Transfus Apher Sci
Roth WK, Busch MP, Schuller A, Ismay S, Cheng A, Seed CR, et al.
International survey on NAT testing of blood donations: Expanding implementation and yield from 1999 to 2009. Vox Sang
Phikulsod S, Oota S, Tirawatnapong T, Sakuldamrongpanich T, Chalermchan W, Louisirirotchanakul S, et al.
One-year experience of nucleic acid technology testing for human immunodeficiency virus type 1, hepatitis C virus, and hepatitis B virus in Thai blood donations. Transfusion
Agarwal N, Chatterjee K, Coshic P, Borgohain M. Nucleic acid testing for blood banks: An experience from a tertiary care centre in New Delhi, India. Transfus Apher Sci
Kumar R, Gupta S, Kaur A, Gupta M. Individual donor-nucleic acid testing for human immunodeficiency virus-1, hepatitis C virus and hepatitis B virus and its role in blood safety. Asian J Transfus Sci
Mangwana S, Bedi N. Significance of nucleic acid testing in window period donations: Revisiting transfusion safety in high prevalence-low resource settings. J Pathol Nepal
Wendel S, Levi JE, Takaoka DT, Silva IC, Castro JP, Torezan-Filho MA, et al.
Primary screening of blood donors by nat testing for HCV-RNA: Development of an “in-house” method and results. Rev Inst Med Trop Sao Paulo
Hourfar MK, Jork C, Schottstedt V, Weber-Schehl M, Brixner V, Busch MP, et al.
Experience of German Red Cross blood donor services with nucleic acid testing: Results of screening more than 30 million blood donations for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus. Transfusion
Mathur A, Shah J, Shah R, Shah P, Harimoorthy V, Choudhury N, et al.
A study on optimization of plasma pool size for viral infectious markers in Indian blood donors using nucleic acid amplification testing. Asian J Transfus Sci
Smith DB, Davidson F, Simmonds P. Hepatitis C virus variants and the role of genotyping. J Hepatol
(Suppl 2) : 26-31.
Scott JD, Gretch DR. Molecular diagnostics of hepatitis C virus infection: A systematic review. JAMA
Singh B. Nucleic Acid Testing (NAT) screening of blood donors in India. A Project Report, International Hospital Federation Reference Book, 2008/2009. Bernex: The International Hospital Federation (IHF); 2009. p. 66-8.
Hans R, Marwaha N. Nucleic acid testing-benefits and constraints. Asian J Transfus Sci
Chandrashekar S. Half a decade of mini-pool nucleic acid testing: Cost-effective way for improving blood safety in India. Asian J Transfus Sci
Weusten J, Vermeulen M, van Drimmelen H, Lelie N. Refinement of a viral transmission risk model for blood donations in seroconversion window phase screened by nucleic acid testing in different pool sizes and repeat test algorithms. Transfusion
[Figure 1], [Figure 2]