Introduction Hepatitis C virus (HCV) is an emerging virus of medical importance. Approximately 3% of the world's population has been infected with the HCV, which represents about 170 million chronic carriers at risk of developing serious complications. The World Health Organization considers Hepatitis C an epidemic, as it can infect a patient for decades before being discovered, & is often called Silent Epidemic.

Source: World Health Organization, HCV status 2002.

Epidemiology of Hepatitis C

A. Prevalence 1. Total Population effected worldwide: 3% 2. Intravenous Drug Abuse: 97% (some communities)

B. Incubation 7-8 weeks 1. HCV RNA found in blood within 3 weeks post-

exposure C. Transmission: by Blood Products and blood exposure

1. Intravenous Drug Abuse 2. Intravenous Immunoglobulin 3. Transfusion

a. HCV accounts for 85% transfusion associated hepatitis

4. Tattoo needles 5. Organ transplant 6. Vertical transmission from mother to child

a. Delivery method does not alter transmission rate

b. Average rate: 6% c. HIV co infection: 17%

7. Needle stick injury (4-10% rate of infectivity) a. Seroconversion in 2200 healthcare workers per

year 8. No apparent parenteral risk factor in 40% of cases

D. Transmission by other body fluid is less common 1. Transmission to simple household contacts is rare 2. No association with Lactation 3. Sexual transmission is much less common

a. Prevalence 1.5% in long term partners b. Higher risk behaviors that raise transmission

i. Multiple partners ii. Early sex iii. Non-Condom use iv. Sex with associated trauma v. Co morbid Sexually Transmitted Disease

THE VIRUS

HCV is a positive, single-stranded RNA virus in the Flaviviridae family. The hepatitis C virus is an enveloped RNA virus with a diameter of about 50 nm, classified as a separate genus (Hepacivirus) within the Flaviviridae family.

Genetic Organization:-

• The HCV genome contains a approximately 9000 nucleotides (nt), flanked by untranslated regions at its 5’ and 3’ extremities

• These nucleotides encode a long polyprotein of 3022 amino

acids. Concomitantly with its translation, it is cleaved by cellular and viral proteases into ten different products, which are, three to four structural proteins, and the rest are the non-structural proteins. Their organization and functions are described below.

1. Core Proteins: - The first structural protein, from the N-

terminus of the polyprotein, is the core protein. It constitutes the virion nucleocapsid.

2. Envelope Proteins: - The next two proteins are the envelope

glycoproteins E1 and E2, are exposed on the virion surface. E2 mediates viral binding to the cells.

3. Non Structural Proteins: - The nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) have various functions involved in viral RNA replication or proteolytic processing of the polyprotein. NS2 and NS3 are the two viral proteases responsible for the cleavage of all the NS proteins. Furthermore, NS3 has a helicase activity and NS4A is a cofactor of NS3. NS5 helps in the replication of the virus

It is estimated that 80- 85% of patients who are infected with the virus will develop chronic infection. This happens despite a healthy immune system. There is no evidence to indicate that most patients with HCV have any immune defect. The virus appears to subtly alter its genetic structure over time and this is one of the theories on how it evades immune clearance. The virus can be found within the liver cell (hepatocyte) and in the blood.

Generally there are low titers of the virus in the serum of infected patients. It is very difficult to grow in cell culture, and no one has ever seen the virus with electron microscopy. A positive HCV antibody test indicates infection with the virus at some stage (past and/or current).

Genotypes and their Distribution

Heterogeneity of genomes is observed between different HCV isolates - a result of the accumulation of mutations during evolution of the viruses. This diversity has led to its classification into ‘genotypes’ & ‘subtypes’, based upon genetic relatedness. The most commonly used classification of Hepatitis C virus has HCV divided into the following genotypes (main types): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. As highlighted, HCV genotypes can be broken down into sub-types, some of which include:

1a, 1b, 1c 2a, 2b, 2c 3a, 3b, 3f, 3g 4a, 4b, 4c, 4d, 4e 5a 6a 7a, 7b 8a, 8b 9a 10a 11a

Although each genotype was initially thought to have a distinct geographical location it is now clear that different genotypes are distributed worldwide (Takada et al 1993). No well-defined culture system or classification based upon clinical manifestations has yet been identified for these variants. These genetic variations are important as they may have an effect on clinical outcome, on response to treatment, on diagnostic testing and on vaccine development. The current general global patterns of genotypes and subtypes:

1a - mostly found in North & South America; and in Australia 1b - mostly found in Europe and Asia. 2a - in Japan and China. 2b - is the most common in the U.S. and Northern Europe. 2c - the most common in Western and Southern Europe. 3a - highly prevalent in Australia and South Asia.

4a - highly prevalent in Egypt 4c - highly prevalent in Central Africa 5a - highly prevalent only in South Africa 6a - restricted to Hong Kong, Macau and Vietnam 7a & 7b - common in Thailand 8a, 8b & 9a - prevalent in Vietnam 10a & 11a - found in Indonesia.

Distribution of HCV subtypes in India:-

Phylogenetic analysis of the sequences of the 149 samples from hepatitis C virus (HCV) positive chronic carriers representing northern, southern, eastern, and western India showed that type 3(a, g & f) and type 1 (a &b) are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4d viruses (6.04%) were seen only in southern India.

REPLICATION The hepatitis C virus must attach to and infect liver cells in order to carry out its life cycle and reproduce - this is why it is associated with liver disease. The life cycle is as follows:

1. The virus locates and attaches itself to a liver cell. Hepatitis C uses particular proteins present on its protective lipid coat to attach to a receptor site (a recognizable structure on the surface of the liver cell).

2. The virus's protein core penetrates the plasma membrane and enters the cell. To accomplish this, hepatitis C utilizes its protective lipid (fatty) coat, merging its lipid coat with the cell’s outer membrane (the coat is in fact composed of a fragment of another liver cell's plasma membrane). Once the lipid coat has successfully fused to the plasma membrane, the membrane engulfs the virus - and the viral core is inside the cell.

3. The protein coat dissolves to release the viral RNA in the cell. This may be accomplished during penetration of the cell membrane (it is broken open when it is released into the cytoplasm), or special enzymes present in liver cells may be used to dissolve the casing.

4. The viral RNA then co-operates with the cell's ribosomes, and begins the production of materials necessary for viral reproduction. Because hepatitis C stores its information in a "sense" strand of RNA, the viral RNA itself can be directly read by the host cell's ribosomes, functioning like the normal mRNA present in the cell. As it begins producing the materials coded in its RNA, the virus also probably shuts down most of the normal functions of the cell, conserving its energy for the production of viral material, although it occasionally appears that hepatitis C will stimulate the cell to reproduce (presumably to create more cells that can produce viruses), which is why hepatitis C is often associated with liver cancer. The viral RNA first synthesizes the RNA transcriptase it will need for reproduction.

5. Once there is adequate RNA transcriptase, the viral RNA creates an antisense version (the paired opposite) of itself as a template for the creation of new viral RNA. The viral RNA is now copied hundreds or thousands of times, making the genetic material for new viruses. Some of this new RNA will contain mutations.

6. Viral RNA then directs the production of protein-based capsomeres (the building blocks for the virus's protective protein coat). Ribosomes create the proteins and release them for use.

7. The completed capsomeres assemble around the new viral RNA into new viral particles. The capsomeres are designed to attract each other and fit together in a certain way. When enough capsomeres are brought together, they self-assemble to form a spherical shell, called a capsid, which fully encapsulates the virus's RNA. The completed particle is called a nucleocapsid.

Life Cycle Of HCV

8. The newly formed viruses travel to the inside portion of the plasma membrane and attach to it, creating a bud. The plasma membrane encircles the virus and then releases it - providing the virus with its protective lipid coat, which it will later use to attach to another liver cell. This process of budding and release of new viruses continues for hours at the cell surface until the cell dies from exhaustion.

Each surviving virus - those that are not destroyed by the immune system or other environmental factors - can produce hundreds or thousands of offspring. Over time, this endless cycle of reproduction results in significant damage to the liver, as millions upon millions of cells are destroyed by viral reproduction or by the immune system's attacks on infected cells.

Clinical Manifestations

Acute Infection

Patients infected with HCV are usually asymptomatic, or have non- specific symptoms such as general fatigue and mild nausea. After an incubation period of about 60 days, acutely infected patients develop elevated serum alanine aminotransferase (ALT) enzyme levels with peak levels typically averaging 600 U/L. Up to 25% of infected patients experience an icteric illness. Symptoms and signs, when they occur, are similar to those of other forms of acute viral hepatitis, but are generally milder than those seen in Hepatitis B infection.

Chronic State

From the available data it is estimated that a high proportion (greater than 70%) of Hepatitis C infections lead to a chronic carrier state which is asymptomatic in most cases. Symptoms, when present, may be non-specific and therefore not obviously connected with the previous acute phase of the illness. Symptoms may include mild to severe fatigue and right upper quadrant abdominal pain or discomfort. Jaundice, fever, chills or night sweats, malaise, concentration problems, headaches and nausea may also occur. Raised levels of liver enzymes (ALT, AST) are common and may persist for years as acute infection enters a chronic phase. However, the levels of these enzymes can fluctuate and may be within the normal range at times. It is believed that the virus circulates in the bloodstream at a low level, sometimes below the present levels of detection. Liver damage, as determined by liver biopsy, does seem to

gradually occur over the years but at any one time ALT levels and virus detection in the serum may not reflect this. It is estimated that 25-30% of individuals with chronic HCV infection will develop cirrhosis over an average of twenty years, linking low-level replication of the virus with liver injury. Hepato-cellular carcinoma and liver failure may develop in a percentage of those that have cirrhosis. †Most people who contract HCV become chronic carriers †many people who are HCV antibody positive will be asymptomatic †chronic carriers are at a higher risk of developing liver damage.

DIAGNOSIS: -

There is a range of tests, which help in the Diagnosis of infection from HCV virus. They are: -

A. LIVER FUNCTION TEST

The following are markers are tested for diagnosis of the infection:

A. Markers of hepatocyte injury 1. Alanine transaminase (ALT)

a. Most specific for hepatocyte injury 2. Aspartate transaminase (AST)

a. Less specific than ALT (present outside liver) b. ALT/AST ratio >2 in Alcoholism and Wilson's Disease

3. Lactate Dehydrogenase (LDH) a. Least specific for hepatocyte injury b. Dramatically increased in ischemic hepatitis c. Increased with alkaline phosphatase in liver

metastases.

These tests are majorly indicating liver injury and may show positive results even in cases where the liver injury is caused by other diseases. Thus are less specific and are rarely used for primary diagnosis.

B. VIRAL LOAD

The quantity of virus in the peripheral blood can be quantified. There is evidence to support the assertion that patients with persistently low levels of virus have a greater chance of responding to antiviral therapy compared to those with persistently high levels of the virus. These tests are difficult to perform and require high level of expertise. Also this test requires specialized equipments and materials, And thus is not practical and is quite expensive.

C. PCR (POLYMERASE CHAIN REACTION)

• A sensitive technique for detecting viral RNA.

• Even small amounts of RNA can be amplified and detected. • Is a marker of viral replication and infectivity if positive. • The commonly available test gives a non-qualitative result (i.e. detected

or not detected).

The PCR test can be very useful, as in the following situations:

• If the anti-HCV and confirmatory test are equivocal and the ALT consistently normal, a negative PCR strongly suggests that the patient does not have hepatitis C.

• If a patient has normal LFTs during Interferon therapy but has positive PCR 6-12 months into therapy, they are likely to relapse when therapy ceases.

• If the anti-HCV test is positive and the ALT consistently normal, and the PCR negative on two occasions over six to 12 months, then the patient has probably recovered after past infection.

If the patient has several possible causes for liver disease and is anti-HCV positive, a positive PCR suggests that HCV is playing a role in the liver disease. But PCR is quite expensive and requires specialized equipments and Instruments. Also his test takes about 4-5 hrs to get completed.

D. ANTI - HCV ANTIBODY

The test for antibodies to HCV is highly valuable in the diagnosis and study of the infection, especially in the early diagnosis of HCV after transfusion.

• A range of products by J. Mitra & Co. Pvt Ltd measures antibody response to virus, Such as:-

A. HCV TRI-DOT.

B.HCV MICROLISA.

Generation of Tests in the Diagnosis of antibodies to specific markers for HCV and their Importance: -

è The first generation anti HCV assay used C100-3 peptide where as the second generation assay used several recombinant viral proteins and peptides typically C-22 from the core region, C33-C

from the second non-structural (NS3) region and 5-1-1 & C100-3 from the NS4 region. They were associated with a high rate of both false positive and false negative results.

è This led to the development of third generation anti-HCV assay which uses a greater range of antigens from core, NS3, NS4 & NS5 regions of the HCV genome, thus providing greater sensitivity and better specificity.

è Recently the 4th generation assay for testing of anti-HCV has been established. The 4th Generation tests as HCV TRI-DOT utilizes a unique combination of modified HCV antigens from the putative core, NS3, NS4 & NS5 regions of the virus to selectively identify all subtypes of Hepatitis C Virus in human serum/plasma with a high degree of sensitivity and specificity. The antigens used are chemically treated and unfolded in a special way to make them more reactive & specific to different epitopes of core & NS3 region thereby minimizing the chances of cross reactivity & enhancing the specificity. Also, the superior sensitivity of the test allows for the significantly earlier detection of antibodies during sero-conversion following HCV infection, thereby reducing the incidence of post transfusion hepatitis and providing a safer blood supply.

HCV TRI-DOT The 4th Generation HCV TRI-DOT is a rapid, visual, sensitive and qualitative in vitro diagnostic test based on Flow Through Technology for the detection of antibodies to Hepatitis C Virus in human serum or plasma.

The 4th Generation HCV TRI-DOT has been developed and designed with increased sensitivity for core and NS3 antibodies using a unique combination of modified HCV antigens. They are for the putative core (structural), protease/helicase NS3 (non-structural), NS4 (non-structural) and replicase NS5 (non-structural) regions of the virus in the form of two test dots “T1” & “T2” to provide a highly sensitive and specific diagnostic test.

HCV MICROLISA

The 3rd generation HCV Microlisa is an in vitro qualitative enzyme linked immunosorbent assay for the detection of antibodies against HCV (anti-HCVs) in human serum or plasma. The kit is basically intended to screen blood donations to identify and eliminate the infected units of blood and for clinical diagnostic testing.

Inferences drawn from the Diagnostic Tests:-

TEST RESULTS INTERPRETATION RECOMMENDATION

Anti-HCV Positive

Chronic hepatitis, chronic hepatitis C recovered, recent acute hepatitis C, or false positive test

Further evaluation

Anti-HCV

Positive

ALT

Normal

EIA Positive

Possible chronic HCV or recovered infection

Further evaluation

Anti-HCV

Positive

ALT

Elevated

EIA Positive

Presume chronic hepatitis C

Further evaluation/ consider Interferon therapy

Anti-HCV

Positive

ALT

Normal

EIA Negative

/indeterminate

Presume false positive anti-HCV or recovered

Further evaluation by HCV-RNA PCR test

Anti-HCV

Positive

ALT

Elevated

Presume false positive anti-HCV, false negative supplemental test unlikely

Further evaluation for liver disease other then hepatitis C + HCV RNA PCR

EIA Negative

ALT (no other +ve tests)

Elevated Other liver diseases Further evaluation

Prevention:-

Comprehensive strategy to prevent and control hepatitis C virus (HCV) infection:-

• Primary prevention activities include - screening and testing of blood, plasma, organ, tissue, and semen donors - virus inactivation of plasma-derived products - adequate sterilization of reusable material such as surgical or dental instruments - risk-reduction counseling and services - implementation and maintenance of infection-control practices - needle and syringe exchange programs

• Secondary prevention activities include - identification, counseling, and testing of persons at risk - medical management of infected persons

• Professional and public education

• Surveillance and research to monitor disease trends and the effectiveness of prevention activities and to develop improved prevention methods.

Prevention of spread of infection should be the main goal at the current time until cost effective therapies become available.

Treatment

The rationales for treatment of chronic hepatitis are to reduce inflammation, to prevent progression to fibrosis, cirrhosis, and HCC through the eradication of the virus in chronically infected patients, and to decrease infectivity and control the spread of the disease.

Combination therapy results in better treatment responses than monotherapy; the highest response rates have been achieved with interferon in combination with ribavirin. Genotype determinations influence treatment decisions. Currently

the best indicator of effective treatment is a sustained viral response, defined by the absence of detectable HCV RNA in the serum.

Interferon has been shown to normalize liver tests, improve hepatic inflammation and reduce viral replication in chronic hepatitis C and is considered the standard therapy for chronic hepatitis C.

Transplantation is an option for patients with cirrhosis who manifest clinically evident end-stage liver disease. After transplantation, however, the donor liver almost always becomes infected, and the risk of progression to cirrhosis reappears.

To protect their liver, people with HCV infection should avoid alcohol consumption, not start any new medicine (not even herbal) without a physician’s knowledge, and get vaccinated against hepatitis A and B.

Response to therapy is influenced by duration of therapy, dosage, infecting viral load and disease stage. The strain of the infecting virus may also affect the clinical response. The treatment strategy to be adopted depends on availability of drugs and cost. Patient adherence is critical to the success of HCV treatment

Glossary

ALT:- alanine aminotransferase, an enzyme that interconverts L-alanine and D-alanine. It is a highly sensitive indicator of hepatocellular damage. When such damage occurs, ALT is released from the liver cells into the bloodstream, resulting in abnormally high serum levels. Normal ALT levels range in man from 10 to 32 U/l; in women, from 9 to 24 U/l. The normal range for infants is twice that of adults.

AST (aspartate aminotransferase) the enzyme that catalyzes the reaction of aspartate with 2-oxoglutarate to give glutamate and oxaloacetate. Its concentration in blood may be raised in liver and heart diseases that are associated with damage to those tissues. Normal AST levels ranges from 8 to 20 U/l. AST levels fluctuate in response to the extent of cellular necrosis.

Bilirubin is the chief pigment of bile, formed mainly from the breakdown of hemoglobin. After formation it is transported in the plasma to the liver to be then excreted in the bile. Elevation of bile in the blood (>30 mg/l) causes jaundice.

Carrier is a person who has HCV in his or her blood even if all symptoms have disappeared. Because the virus is present in the blood, it can be transmitted to others.

Cirrhosis a chronic disease of the liver characterized by nodular regeneration of hepatocytes and diffuse fibrosis. It is caused by parenchymal necrosis followed by nodular proliferation of the surviving hepatocytes. The regenerating nodules and accompanying fibrosis interfere with blood flow through the liver and result in portal hypertension, hepatic insufficiency, and jaundice. Cirrhosis is a more severe, irreversible process of liver inflammation, necrosis, and regeneration. In hepatitis C, cirrhosis occurs as a late stage sequel of chronic infection, and may take 20-30 years to develop.

ELISA enzyme-linked immunoassorbent assay

Endemic continuously prevalent in some degree in a community or region.

Endoplasmic reticulum a network or system of folded membranes and interconnecting tubules distributed within the cytoplasm of eukaryotic cells. The membranes form enclosed or semi enclosed spaces. The endoplasmic reticulum functions in storage and transport, and as a point of attachment of ribosomes during protein synthesis.

Epidemic is an outbreak of disease such that for a limited period a significantly greater number of persons in a community or region suffer from it than is normally the case. Thus an epidemic is a temporary increase in incidence. Its extent and duration are determined by the interaction of such variables as the nature and infectivity of the casual agent, its mode of transmission and the degree of preexisting and newly acquired immunity.

Genotype:- the genetic constitution of an individual.

Hepatocytes are liver cells.

Histopathology the study of the structural alterations of cells and tissues caused by disease.

Humoral: pertaining to the humors, or certain fluids, of the body.

Incidence the number of cases of a disease, abnormality, accident, etc., arising in a defined population during a stated period, expressed as x cases per 1000 persons per year.

Interferon a protein produced in organisms infected by viruses, and effective at protecting those organisms from other virus infections. Interferons exert virus-

nonspecific but host-specific anti viral activity by inducing the transcription of cellular genes coding for anti viral proteins that selectively inhibits the synthesis of viral DNA and proteins. Interferons also have immuno-regulatory functions. Production of interferon can be stimulated by viral infection, especially by the presence of double stranded RNA, by intracellular parasites, by protozoa, and by bacteria and bacterial products. Interferons have been divided into three distinct types associated with specific producer cells and functions, but all animal cells are capable of producing interferons, and certain producer cells (leukocytes and fibroblasts) produce more than one type (both a and ß).

Jaundice is a yellow discoloration of the skin and mucous membranes due to excess of Bilirubin in the blood, also known as icterus.

Lymphocyte:- a leukocyte of blood, bone marrow and lymphatic tissue. Lymphocytes play a major role in both cellular and humoral immunity, and thus several different functional and morphologic types must be recognized, i.e. the small, large, B-, and T-lymphocytes, with further morphologic distinction being made among the B-lymphocytes.

Prevalence is the number of instances of infections or of persons ill, or of any other event such as accidents, in a specified population, without any distinction between new and old cases.

Prophylaxis is the prevention of disease, or the preventive treatment of a recurrent disorder.

Reverse transcriptase is an enzyme that catalyzes the formation of DNA using an RNA template, and is thus an RNA-dependent DNA polymerase. The name refers to the fact that the enzyme transcribes nucleic acids in the reverse order from the usual DNA-to-RNA transcription. RIBA™ recombinant immunoblot assay

RT-PCR reverse transcriptase - polymerase chain reaction. A technique commonly employed in molecular genetics through which it is possible to produce copies of DNA sequences rapidly. Qualitative RT-PCR for HCV test to detect HCV RNA by amplification of viral genetic sequences. Quantitative assays for HCV RNA tests to detect HCV RNA concentration (viral load) by amplification of viral genetic sequences or by signal amplification.

Seroconversion:- the production in a host of specific antibodies as a result of infection or immunization. The antibodies can be detected in the host’s blood serum following, but not preceding, infection or immunization.

Serotype a subgroup within a species, defined by reaction of one or more antigens with the corresponding anti serum.

Serum is the clear, slightly yellow fluid, which separates from blood when it clots. Sera containing antibodies and antitoxins against infections and toxins of various kinds (antisera) have been used extensively in prevention or treatment of various diseases.

Viremia:- the presence of viruses in the blood, usually characterized by malaise, fever, and aching of the back and extremities.

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