A And E College of Pharmacy, Baluahi, Mohiuddin Nagar Samastipur, Bihar.
Viral hepatitis is a major public health concern. It is associated with life threatening conditions including liver cirrhosis and hepatocellular carcinoma. Hepatitis C virus infects around 71 million people annually, resultantly 700,000 deaths worldwide. Extrahepatic associated chronic hepatitis C This review included a total of 150 studies that revealed almost 19 million people are infected with hepatitis C virus and 240,000 new cases are being reported each year. This trend is continually rising in developing countries like Pakistan where intravenous drug abuse, street barbers, unsafe blood transfusions, use of unsterilized surgical instruments and recycled syringes plays a major role in virus transmission. Almost 123–180 million people are found to be hepatitis C virus infected or carrier that accounts for 2%–3% of world’s population. The general symptoms of hepatitis C virus infection include fatigue, jaundice, dark urine, anorexia, fever malaise, nausea and constipation varying on severity and chronicity of infection.
In the United States, hepatitis C virus (HCV) infection is a leading cause of liver-related deaths, cirrhosis, and hepatocellular carcinoma.1 HCV was discovered in 1989, but it was not until 1992 that the blood supply was screened for HCV; therefore, prior to 1992, contaminated blood products had been a primary cause of infection. According to the etymology of word the “hepatitis,” it is a Latin word that refers to the swelling of hepatic tissues. Currently, viral hepatitis is considered as a major public health concern that is threatening mankind at major masses especially in the Asian developing countries like Pakistan. The current approved therapeutic protocols for the treatment of chronic hepatitis C virus (HCV) infection issued by the Infectious Diseases Society of America (IDSA) and the American Association for the Study of Liver Diseases (AASLD)—titled “the HCV guidance”—involve combining various DAAs. A major obstacle to the sustained success of DAAs as a curative treatment for HCV, let alone HCV eradication, is the emergence of HCV variants resistant to DAAs.2The presence of resistance-associated substitutions (RASs), which can occur naturally or develop during treatment, has the potential to reduce the effectiveness of treatment, with the possibility of forward transmission of drug-resistant variants, especially within high-risk populations.
2.History of hepatitis c
It became evident by the mid-1970s that a viral hepatitis agent, distinct from hepatitis A virus (HAV) and hepatitis B virus (HBV), was responsible for post-transfusion hepatitis, and this putative clinical entity was termed “non-A, non-B” hepatitis (NANBH) 3 Subsequent investigation involving transfusion recipients revealed that acute clinical manifestations of NANBH were generally less severe than those of hepatitis; nevertheless, NANBH was capable of leading to grave outcomes, including cirrhosis and hepatic failure. Progress was made through the serial passage of NANBH in chimpanzees, which provided critical pathological, physiological, and biochemical insights, alongside a valuable number of specimens for further study.4 A breakthrough was achieved by the Michael Houghton team, which constructed a lambda phage library from cDNA of a high-titer chimpanzee plasma specimen. The Michael Houghton team screened more than 1 million expression clones with serum from a chronic NANBH patient, which led to identification of a single positive cDNA clone, designated 5-1-1, marking the discovery of HCV.This led to the development of initial assays for detecting anti-HCV antibodies, incorporating the 5-1-1 antigen.5 Despite advancements in screening and the prevention of hepatitis C, it remains a significant global health concern.
3.Hcv origin and classification
HCV is a member of the Hepaciviral genus within the Flaviviridae family, a classification delineated through seminal studies led by Peter Simmonds.6 A notable review conducted by Peter Simmonds meticulously examined the complex narrative surrounding the origins and evolutionary trajectory of HCV. The aforementioned review highlighted the challenges to pinpoint the ancient history of HCV in human populations and recommended a cautious interpretation of the term “origin”, particularly in reference to HCV global dissemination and diversification during the 20th century.7 In exploring the genetic heterogeneity of HCV, phylogenetic studies revealed a remarkable divergence within the virus, categorizing HCV into at least seven (or even eight) distinct genotypes, denoted by Arabic numerals.8
Figure 1. The evolutionary history of HCV genotypes (GTs) based on (Sallam, 2017).
The phylogeny was constructed using the neighbor-joining (NJ) method with bootstrap test for evaluation of topology (1000 replicates). Internal branches with bootstrap values ≥ 0.9 are highlighted in black. The evolutionary distances were computed using the TN93 method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 4). The analysis involved 147 NS5B sequences (1495 bases) downloaded from the Los Alamos Hepatitis C sequence and immunology databases accessed on 30 April 2024). Evolutionary analyses were conducted in MEGA6.9 This genetic diversity extends within genotypes, which resulted in further subclassification of these genotypes into several subtypes identified by lowercase English letters. these genotype and subtype classifications are based on variations in nucleotide sequences, with inter-genotypic differences exceeding 30% and intra-genotypic differences ranging between 20 and 25%. Investigation of the evolutionary rate of HCV revealed a relatively rapid rate, estimated at 1.0–2.0 × 10−3 substitutions per site per year (s/s/y), comparable to other RNA viruses.10
4.Hcv genome
The details of the HCV genomic regions can be further explained as follows. Positioned at the beginning of the HCV viral genome is the highly conserved 5′ UTR, which harbors an internal ribosomal entry site (IRES) facilitating the cap-independent translation of the viral genome. This region precedes the coding sequences for structural proteins, including the core protein (C), the envelope glycoproteins (E1 and E2), and the ion-channel verprolin (p7). Notably, the C protein maintains a conserved sequence, whereas E1 and E2 glycoproteins exhibit significant sequence variability, a phenomenon attributed to the pressure of immune selection.11 The HCV genome’s last segment encodes NS proteins, essential for viral replication and processing of the polyprotein. The NS2 protein, a cysteine protease, facilitates the cleavage of NS3 from the NS2–NS3 junction. NS3, together with NS4A, constitutes a serine protease complex essential for the cleavage of subsequent NS proteins, regulating the sequential processing of the viral proteome. NS4B serves as a membrane anchor for the viral replication complex, while NS5A, alongside NS4B, contributes to the formation of the endoplasmic reticulum (ER) membranous web, a structure essential for viral replication dynamics.12
Figure 2. Schematic illustration of HCV genome and polyprotein.
5.Life cycle of Hcv
The percentage of HCV positive cells found in sick liver tissue varies from less than 5%–100%. This can be correlated to virions generation rate of 50 units per hepatocytes per day. players is also able to replicate inside the secondary mononuclear cells of blood.13
Fig 3. Graphical depiction of HCV life cycle
5.1. Attachment and cell entry
HCV life cycle begins when the infectious particle attaches to host cell and explicit in vitro interaction between CD81 receptor (located superficially on the host cell) and the viral attachment protein (E2 glycoprotein on the outside of the particle). CD81 has been recognized receptor for other viral particles as well. This interaction is really an essential step for a virus to start an infection. For penetration into the cell, HCV needs to attach to the low-density lipoprotein (LDL) receptors. E1 is implicated inside the union of membrane.14
5.2. Polyprotein translation and processing
The translation of the genomic DNA is instantly started as it sets foot in the cytoplasm. RNA translation is arbitrated by internal ribosome entry sites (IRES) rather than by a Cap-dependent method. There are various aspects which influence the function of HCV IRES. First, the IRES-dependent translation of the X-Tail is accomplished which is present at the farthest 3′end of the HCV genome. Second, to activate the translation, several cell features bind to the IRES including polypyrimidine-tract-binding (PTB) protein, the La antigen, heterogeneous nuclear ribonucleoprotein L and other unknown proteins.
5.3. RNA replication
The synthesis of minus (–) and plus (+) strand RNA is primarily catalyzed by NS5B RNA dependent RNA polymerase (RdRp). A 3′ end is produced by intramolecular back folding or hybridization of the sequences at the 3′ end which is exploited for the elongation. NS5B can produce RNA primer due to the elevated concentrations of the GTP or ATP. Full-length genome of HCV is replicated by NS5B in vitro. However, other viral or cellular factors are also mandatory in vivo. The NS3 helicase is a likely viral candidate that keeps RNA template stable and aid in duplication of the NS5A phosphoprotein implicated for the management of RNA replication. Furthermore, PTB collaborates with the sequences present at the 3′ non-translated region (NTR). Glyceraldehyde-3-phosphate dehydrogenase (G3P dehydrogenase), interacts with the poly (U)-sequence in the 3′ NTR and the p87 and p130 cellular proteins. HCV replication might also be obstructed by proteins from other viruses.15 for instance elevated load of Epstein bar virus (EBV). This happens probably due to the stimulation of transcription of the cellular genes. Owing to its pivotal role in viral RNA synthesis, the NS5B polymerase has been a prime target for antiviral development.
6.Pathological consequences of HCV infection
The HCV infection results in several pathological conditions in the patients depending upon their immune capability.
6.1 Steatosis
Patients suffering from chronic HCV often suffer from a histological feature called as liver steatosis. Also known as fatty liver disease, this condition is characterized by too much fat build up in the liver. Two major factors, that is, genetic and epigenetic, play a contributing role in the developing link between hepatic steatosis and HCV. HCV can alter the intrahepatic metabolism of lipid by affecting lipid peroxidation, lipid synthesis, insulin resistance, assembly and secretion of very low-density lipoprotein (VLDL) and oxidative stress. The host-mediated and viral factors serve as major contributing factors for the hepatic steatosis buildup being overweight, diabetes mellitus, insulin resistance, alcohol consumption, and hyperlipidemia.16
6.2 Fibrosis
Liver fibrosis is the development of imprudent fibrous connective tissue that comprises extra cellular matrix proteins such as collagen fibers emitted by the activate hepatic stellate cells.180 It is mediated by wound healing response in response to due to tissue damage by chronic HCV infection. ever fibrosis is a considerable complication of HCV infection, and its continuation can cause life threatening conditions such as liver failure, LC and hepatocellular carcinoma. For the treatment of liver fibrosis, viral eradication can contribute to decrease the liver damage by ameliorating the inflammation process and retrogressing the fibrosis regardless of the treatment method the fibrosis posttreatment with sustained virological response (SVR) still needs to be determined. 17
6.3 Cirrhosis
LC is the development of a censorious period during chronic liver disease caused by HCV. Without the provision of antiviral therapy, nearly 67%–91% of the patients have to face death because of other liver disorders such as liver function failure and liver cancer. Cirrhosis is a condition in which the normal liver structure exhibits disruption resulting from fibrosis and a nodule is generated that obstruct the normal functioning of liver. Old age, chronic HCV infection and excessive alcoholism serves as the risk factors for cirrhosis. After onset of HCV infection, cirrhosis takes almost 30 years on an average to develop. This average also significantly varies from person to person. Almost 4% annual deaths worldwide are caused by cirrhosis. It is estimated that Pakistan has the second highest HCV prevalence. Patients suffering from cirrhosis and hypertension have 30% risk of developing hemorrhaged and bleeding conditions.18
6.4 Hepatocellular carcinoma
Hepatocellular Carcinoma (HCC) is a heterogeneous and most common malignant tumor group that varies in genetic and epigenetic alteration events and risk factors. HCC is considered as the most frequent cause of primary liver malignancy and a main cause for worldwide cancer-related death. HCC is the ninth leading cause of deaths in United States. In last 15 years, there is an increase in the mortality rate linked with HCC. Incidence and mortality associated with HCC continue to increase despite of the advancements in prevention, screening, diagnostic, and treatment methods. Regardless of ethology, cirrhosis plays an important role as being a significant risk factor for HCC development. Male population is more vulnerable to cure HCC than female population.19
7.Immunopathogenesis of Hcv
Fig. 4. Immunopathogenesis of HCV
An effective immune response toward HCV infection is hindered by multitude of viral proteins including core, E2, and NS5A protein. Both the innate and adaptive immunity play role in immunopathogenesis of HCV.
7.1 Innate immune response against HCV
The activation of innate immunity in reaction to the HCV invasion plays a crucial role in controlling the virus spreading. It causes apoptosis of hepatocyte which controls the virus progression. In addition, it induces the adaptive immunity as well. In acute HCV infections, the human cytoplasm contains the viral RNA genome. Following the virion’s uncoating, intracellular RNA genome of the virion induces the production of Toll-like receptor 3 (TLR3), Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) inside the infected liver cells. This leads to the production of Type 1 Interferon (IFN-I, α and β) and IFN-γ. In vitro research has revealed that IFN generated by NK cells suppresses HCV replication directly.20
7.2 Adaptive immune response against HCV infection
The key mechanism of viremia regulation in the adaptive immune system is T cell response. HCV-infected hepatocytes are destroyed by specialized CD8+ T lymphocytes through human leukocyte antigen (HLA) class I antigen presentation cells and by cytokine production (TNF-α, IFN-γ). This.occurs when IL-2 stimulates NK cell and CD8+ T cell activation, which is supported by helper CD4+ T cell. Viremia lasting for 6 months or more, is considered persistent HCV infection. HCV uses a variety of methods to evade the immune system, resulting in immune system evasion and infection persistence. T cell function is lost in chronic T cell stimulation, which is the first main mechanism of chronic infection. HCV-specific CD4 + helper T cells produce less IL-2 during the persistent infection, hindering activation of CD8+ T cell.21
7.3 Immune system and chronic HCV infection
In case of chronic HCV infection, the activity of CD4+ T cells is essential. In acute infection of HCV, a robust CD4+ T cell response is linked to viral clearance. However, the decline of the CD4+ T cell activity specific to HCV is closely linked to the transition of acute infection of HCV into the chronic HCV infection. Moreover, inadequacy and the subsequent reduction in a strong CD4+ T cell activity following acute infection have been documented and linked to chronic infection. HCV escape mutations for individuals having multiple HLA epitopes (plus the HLA-DRB1*15 epitope) have been hypothesized as one route which decreases the CD4+ T cell activity. Virus epitopes targeted by CD8+ T lymphocytes are mutated in 50%–70% of the individuals with persistent HCV infections.22
8.Transmission and risk factors
8.1 Barbers
Barbers are identified as the most likely source of HCV transmission. Inadequate barber hygiene practices can spread HCV to clients. Razor blades can sustain the virus for a few days. Unsterile blades and razors, contaminated with virus-containing blood can profoundly transmit it to another person. Barbers are declared as the prime risk in HCV dispersal by a number of researchers. Comparing the level of awareness between rural and urban regions, it was found that acquaintance and knowledge was about 92% in urban regions whereas it was 68% in rural regions. 23
8.2 Recycled syringes
In the developing countries, reuse of utilized medical syringes in dispensaries, health centers, and healthcare workers is more frequent among families and people with low socioeconomic position. The children of such families are usually involved in the marketing of recycling junk and hospital waste and are at an upraised threat of infections. Among medical care items which can be recycled, therapeutic injections are the second highest. 24
8.3 Intravenous drug users
Human socialization in Pakistan and several other developing countries have made intravenous drug use as the prime risk factor to disseminate this viral disease.
8.4 Transfusion of contaminated blood products
Blood borne pathogen’s dissemination is mediated by the two main risk factors which are blood donation and transfusion. Literature also proficiently considers it as a threat factor for HCV.
8.5 Sexual transmission
Sexual means can also transmit viral hepatitis. Unprotected sex and sexual affairs with multiple partners are the major cause of spread. Majority of the studies performed in Pakistan have concluded it a well-recognized and pronounced mode of transmission.
8.6 Ear and nose piercing
Blood borne pathogens are disseminated via the activities that could lead to blood wounds or seepage. In developing countries, females are more tend toward ear and nose piercings. Therefore, they are at a greater risk for imparting the disease. Usually, unsterilized instruments are being used by the people who do ear and nose piercings. A study had reported 11.7% occurrence of hepatitis due to ear and nose piercings. 25
8.7 Healthcare workers
Viral hepatitis is detected in all the population groups, but it predominates in a few peculiar groups, which are known as the high-risk groups.
8.8 Surgical procedures
Dental surgeries integrate techniques that are prone to needle stick wounds and incur a high probability of the blood infections. Dental surgeries also involve procedures, for instance, use of unsterilized tools that can aid in disease spread.
8.9 Vaccinators
As vaccinators are engaged in a lot of vaccination projects which comprise the employment of injections, they could be a possible risk factor for viral dispersal. Occasionally, in the course of vaccination, virus dispersal can happen from contaminated to uninfected individuals.26
8.10 Perinatal transmission
Transmission of blood borne pathogens occur in the procedures like child delivery as the interior organs are exposed which makes a person more susceptible to various infections.
9. Symptoms of HCV
Fig.5 Symptoms of HCV
9.1 The prodromal phase
Some patients feel sickness, which includes fever, arthralgia, arthritis, rashes, and angioneurotic enema before the proper disease development. These symptoms end before jaundice, which is the most common and peculiar symptom of HCV.
9.2 pre-icteric phase
In this phase, the patient develops respiratory problems and gastrointestinal tract disorders which may include malaise, fatigue, myalgia, nausea, and vomiting, which may be escorted by weight loss, headache, coryza, fever, or pharyngitis and cough. The pre-icteric phase lasts from 2–3 days to 2–3 weeks.
9.3 Icteric phase
Patients develop gastric pain, right upper quadrant discomfort, or diarrhea in the icteric phase. Darkening of urine and light-colored stool are observed in victims.
Table 1. Classifies specific and non-specific symptoms of HCV in icteric phase.
|
Non-specific symptoms |
Specific symptoms |
|
Flu |
Fatigue |
|
Fever |
Jaundice |
|
Arthralgia, rash |
Dark urine |
|
Arthritis |
Lack of appetite |
|
Angio-neurotic |
Bruising or bleeding |
|
Enema |
Vomiting or nausea |
|
|
Liver failure |
10.Diagnosis of HCV
Fig.6 diagnosis of hepatitis c
10.1Serological assay
After the detection of antibodies, further confirmation of the virus should be done with the help of an HCV RNA test. The most common examples of serological assays are:
Screening Tests for anti-HCV. Its common example is Enzyme Immunoassay (EIA)
Supplemental Tests. Example: Recombinant Immune Blot Assay (RIBA)
For the detection of anti-HCV, three generations of tests have been developed up till now and each one is more advanced and sensitive than the previous one. Antigens from the HCV core, non structural (NS) 3, NS4, and NS5 genes are involved in Enzyme Immunoassay 3 and Recombinant Immune Blot
10.2 Molecular assay
The most reliable method of HCV detection is to use Recombinant Immune Blot Assay (PCR) to detect HCV nucleic acid (RNA) in the patient’s plasma or serum. It is well established that qualitative assays are more sensitive than quantitative assays. With sensitivities of 10–50 IU/mL, PCR and Recombinant Immune Blot Assay ((TMA) assays have rendered qualitative assays simpler and more precise. The most sensitive HCV PCR assay currently available has a sensitivity of fewer than 100 copies of HCV RNA per milliliter of plasma or serum. The two main methods for determining HCV RNA levels are discussed here.
10.3Qualitative HCV RNA
The qualitative HCV RNA tests give an all or none answer, indicating whether or not the virus is present in the patient’s body. The amount of virus in the patient’s body is not indicated by this test.
10.4 Quantitative HCV RNA
The quantitative HCV RNA test determines how much HCV is present in the body. This test will also tell you whether your infection is acute or chronic.
10.5 Rationale of screening and molecular tests
The identification of antibodies against HCV in a patient’s blood is the most widely used test for HCV, but the findings may be ambiguous and require careful interpretation. If antibodies against the HCV are present, it indicates that the individual is a chronic HCV carrier (75%–85%), has been infected in the past but the infection has subsided (15%–25%), or has been recently (acutely) infected. After HCV infection, the body needs at least 6–8 weeks to form enough antibodies to be tested in a screening test. Negative HCV antibody test results have a high degree of accuracy. IDUs and people who are involved in other high-risk activities should, however, be retested every year to account for the 6-month window phase.27
11.Treatment
Patients with chronic hepatitis C are given antiviral therapy except for those patients who have co-morbidities. Treatment for HCV is increasingly improving and is successful.
11.1 Pretreatment assessment of patients
Questions regarding the patient’s life after antiviral therapy, as well as other factors such as the duration of infection, signs, and symptoms of disease, and the existence of cofactors that can intensify disease (e.g. alcohol, obesity, co-infections), are asked before treatment. To confirm the amount of HCV RNA and its genotype, pretreatment tests are done, and these tests involve liver biochemistry and function, abdominal ultrasound, fibrosis stage assessment, and tests to rule out co-infections.
11.2 Treatment routines
Fig.7 Treatment routine of HCV
Patients who have never received HCV medication are treated for different periods of time in weeks, depending on the genotype of the HCV.
11.3 post-treatment
Patients who do not reveal any more signs and symptoms of the virus do not require post-treatment, although those with alanine aminotransferase elevation or constant risk exposures (e.g. people who inject drugs) should have annual HCV RNA testing. Patients with cirrhosis and who have had a viral response should be screened for hepatocellular carcinoma regularly. Cirrhosis patients need hepatocellular carcinoma with biannual ultrasound before treatment. Rescue treatment should be provided to patients who have not responded to the viral treatment. Patients who do not get a viral response due to adherence problems or drug-drug interactions should be treated with caution. For 12 weeks, a single-tablet regimen of sofosbuvir, valuative, and vexillaries is effective against all genotypes of HCV.28
12.The future of HCV therapy: high hopes and challenges
Since type I IFN was tested and shown to be effective for treating non-A, non-B hepatitis, the standard of care steadily improved from single-digit success rates to overall virologic cure rates of around 50% with peg-IFN-α and ribavirin. These remarkable advances were achieved empirically, through testing in the clinic, without understanding of how these drugs exerted their anti-HCV effects. So where does the field stand in terms of clinical development? Initial strategies have examined combination of one or more DAAs with peg-IFN-α and ribavirin. This might include one DAA with a high barrier to resistance or two DAAs with lower resistance barriers. Benefits include the direct action of a DAA with the proven and probably independent antiviral activities of peg-IFN-α and ribavirin. This might shorten treatment, particularly for patients with a favorable IFN-λ genotype. Responses are generally superior for HCV genotype 1b versus 1a, for the reasons discussed earlier. However, this strategy also has the potential of increasing the already harsh side effects (as seen for telaprevir and boceprevir) and, in some regimens, is less effective in previous IFN no responders or unusable in patients with contraindications to IFN. Phase 3 studies combining the second-wave protease inhibitors improver or faldaprevir with peg-IFN-α and ribavirin improved SVR rates to ∼80% with milder side effects. Even higher SVR rates of ∼90% were achieved for the nucleotide inhibitor sofosbuvir with peg-IFN-α and ribavirin. Very high SVR rates were also observed with two DAAs (quadruple therapy), either protease and NS5A inhibitors (aquaresis plus daclatasvir or verprolin (GS-9451) plus ledipasvir (GS-5885), or protease and nucleoside polymerase inhibitors (depriver plus emtricitabine (RG7128).
CONCLUSION
HCV infection is a complex systemic disease with serious medical and economic consequences. It is important to assess the full range of HCV disease burden to fully comprehend its impact on patients and the general public. For instance, considering Pakistan as a developing country representative, it is experiencing a historic HCV epidemic, with one out of every 20 citizens previously being infected with the disease which is placing a significant burden on the country economics and healthcare settings. A rapid increase in HCV seroprevalence among the individuals who had previous surgical and medicinal treatments, suggests a major role of hospital-acquired infections in the spread of HCV. Some of the main causative risk factors include needle pricking, barber shaving, blood and its products, dental procedures, IDUs, unsafe delivery methods, dialysis, and vertical transmission from mother to baby. Although efforts are being made to increase the coverage of safe injections, blood examinations, advanced infection management, and assurance of prevention and safe practices in all sectors of healthcare organizations must be assured to accomplish the HCV elimination goal by 2030. However, there is still a long way to go before this global health burden is alleviated and HCV is eradicated. Meanwhile, of the mankind is struggling to achieve this without a prophylactic vaccine. Reduced drug cost, improved access to medication, and most importantly, treatment uptake is also pivotal in combating this disease.
REFERENCE
Supriya Kumari*, Sana Nusrat Praween, Development of analytical method for drugs used in the treatment of hepatitis C, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 9, 3269-3282 https://doi.org/10.5281/zenodo.17222956
10.5281/zenodo.17222956
