Finalizing HCV

8/4/2019 Finalizing HCV

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Muhammad Ali Minhas HCV

Hepatitis C Virus

(HCV)

Introduction:Hepatitis is a disease characterized by inflammation of the liver, usually producing

swelling and, in many cases, permanent damage to liver tissues. A number of different agentscan cause hepatitis, including infectious diseases, chemical poisons, drugs and alcohol. Viralhepatitis refers to a set of at least six viruses that are known to cause hepatitis: hepatitis A(HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), andhepatitis G (HGV). Recent scientific evidence also suggests the existence of other, as yetunidentified hepatitis viruses.

The most common types of viral hepatitis are hepatitis A, B, and hepatitis C. Both hepatitis Band C can lead to serious, permanent liver damage, and in many cases, death.

There are two primary types of viral hepatitis, food-borne and blood-borne hepatitis. The former,which is spread through contaminated food and water, does not cause chronic liver disease. Bycontrast, blood borne viral hepatitis may lead to long-term, persistent infections and chronic liverdisease that has lethal consequences many years after infection.

The Hepatitis C Virus:Hepatitis C is an RNA virus - which means that it mutates frequently. Once an infection

has begun, hepatitis C creates different genetic variations of itself within the body of the host.The mutated forms are frequently different enough from their ancestors that the immune systemcannot recognize them. Thus, even if the immune system begins to succeed against one variation,the mutant strains quickly take over and become new, predominant strains. As a result, thedevelopment of antibodies against HCV does not produce immunity against the disease like itdoes with most other viruses. More than 80% of the individuals infected with HCV will progressto a chronic form of the disease.

There are six basic genotypes of HCV, with 15 recorded subtypes, which vary inprevalence in different regions of the world. Each of these major genotypes can differsignificantly in their biological effects - in terms of replication, mutation rates, type and severityof liver damage, and detection and treatment options. However, these differences are not yetclearly understood.

The 21 current variations in genotype, complicated by the constant mutation of the viruswithin infected individuals, represents a major challenge for the development of treatments and


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vaccinesagainst HCV- and evenfor reliabledetection of

the virus.There is noguaranteethat atreatment,test, orvaccineagainst onestrain will beeffectiveagainst all of

them.Moreover, individuals cured of one strain will be prone to reinfection by any of the other strains.

Anatomy of the Hepatitis C Virus:The structure of the hepatitis C virus is like that of most complex viruses - a core of

genetic material (RNA), surrounded by a protective shell of protein, and further encased in alipid (fatty) envelope of celluar material. However, the fact that the genetic information of thevirus is stored in RNA, not DNA, has important consequences in the life cycle of the virus, andgives hepatitis C its dangerous ability to mutate.

All organisms, with the exception of the RNA viruses, store their permanent informationin DNA, using RNA only as a temporary messenger for information. DNA is quite a stablemolecule, not particularly reactive with other molecules, and the processes which reproduce itmake very few mistakes in the process of copying the molecule (between one in 1 million and 1in 10 million). Most of these mistakes are normally corrected even when they do occur. Thismakes DNA an ideal format for the storage of information, for mutations (errors) only rarelyoccur, and most are not significant.

RNA, by contrast, is a quite reactive molecule, capable of reacting even with itself underthe correct conditions. It also makes frequent mistakes during copying - averaging one mistakeper 10,000 nucleotides each time it is copied. These properties make RNA very poorly suited for

the storage of information.

However, these very propeties make RNA ideal for the storage of viral information. Oncethe immune system has learned to recognize an infecting virus and create antibodies against it(developed an immunity), it can quickly destroy it, so the virus can no longer use that host forreproduction. In order to reinfect a host - it must first change its nature enough that the immunesystem will no longer recognize it - in other words, it must mutate.


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Hepatitis C virus. Structure of the viral capsid is clearly visible

The unstable nature of the RNA molecule provides this mutagenic factor, allowing theHepatitis C virus to develop new genetic variations of itself. As discussed earlier, the mutatedforms are frequently different enough from their ancestors that the immune system cannotrecognize them, so if the immune system begins to succeed against one variation, the mutantstrains quickly take over and become new, predominant strains. Because each surviving virusreproduces itself thousands of times, mutations in the RNA sequence occur frequently, allowingit to evolve faster than any other type of living organism. This evolution is known as antigenicdrift. Mutations occur randomly across the entire length of the viral RNA, and so of course mostare not beneficial, producing viruses which lack a needed protein or are otherwise disadvantaged.However, because of the enormous number of offspring produced by each virus, even a high rateof mutation does not threaten the survival of the virus - and when advantageous mutations dooccur, they are rapidly selected for and reproduced.

Hepatitis C, as an RNA virus, has apowerful reproductive strategy. Because itstores its information in a "sense" strand ofRNA, the viral RNA itself can be directlyread by the host cell's ribosomes,functioning like the normal mRNA presentin the cell. The virus thus needs no specialabilities of its own - it uses the cell's ownribosomes to produce everything it needsfor its takeover of the cell's processes andreproduction. This means hepatitis C

requires only a small amount of RNA to encode its core information, and thus has lots of room


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for genetic variation within the non-essential portions of its RNA. This also gives it fewercommon characteristics that can be readily identified by the immune system - or, for that matter,exploited by scientists working to create a treatment.

Genome of HCV:Hepatitis C virus has a positive sense single-stranded RNA genome. The genome consists

of a single open reading frame that is 9600 nucleotide bases long. This single open reading frameis translated to produce a single protein product, which is then further processed to producesmaller active proteins.

At the 5' and 3' ends of the RNA are the UTR, which are not translated into proteins butare important to translation and replication of the viral RNA. The 5' UTR has a ribosome bindingsite (IRES - Internal ribosome entry site) that starts the translation of a very long proteincontaining about 3,000 amino acids. This large pre-protein is later cut by cellular and viral

proteases into the 10 smaller proteins that allow viral replication within the host cell, or assembleinto the mature viral particles.


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Structural proteins made by the hepatitis C virus include Core protein, E1 and E2; nonstructuralproteins include NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B.

The Life Cycle of Hepatitis C:1) 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. While little isknown about the exact natural processes of hepatitis C, like other viruses, it mustcomplete eight key steps to carry out its life cycle:

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

3) The virus's protein core penetrates the plasma membrane and enters the cell. Toaccomplish this, hepatitis C utlilizes its protective lipid (fatty) coat, merging its lipidcoat with the cells outer membrane (the coat is in fact composed of a fragment ofanother liver cell's plasma membrane). Once the lipid coat has successfully fused tothe plasma membrane, the membrane engulfs the virus - and the viral core is insidethe cell.

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

5) The viral RNA then coopts the cell's ribosomes, and begins the production ofmaterials necessary for viral reproduction. Because hepatitis C stores its informationin a "sense" strand of RNA, the viral RNA itself can be directly read by the host cell'sribosomes, functioning like the normal mRNA present in the cell. As it beginsproducing 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 viralmaterial,

6) 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 Cis often associated with liver cancer. The viral RNA first synthesizes the RNAtranscriptase it will need for reproduction.

7) 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. Theviral RNA is now copied hundreds or thousands of times, making the genetic materialfor new viruses. Some of this new RNA will contain mutations.

8) Viral RNA then directs the production of protein-based Capsomeres (the buildingblocks for the virus's protective protein coat). Ribosomes create the proteins andrelease them for use.

9) The completed Capsomeres assemble around the new viral RNA into new viralparticles. The Capsomeres are designed to attract each other and fit together in acertain way. When enough Capsomeres are brought together, they self-assemble toform a spherical shell, called a capsid that fully encapsulates the virus's RNA. Thecompleted particle is called a nucleocapsid.


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10)The newly formed viruses travel to the inside portion of the plasma membrane andattach to it, creating a bud. The plasma membrane encircles the virus and thenreleases it - providing the virus with its protective lipid coat, which it will later use toattach 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 which are not destroyed by the immune system or otherenvironmental factors - can produce hundreds or thousands of offspring. Over time, this endlesscycle of reproduction results in significant damage to the liver, as millions upon millions of cellsare destroyed by viral reproduction or by the immune system's attacks on infected cells.


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Cellular immune responses against hepatitis C virusHCV, like other viruses,

induces multiple immuneeffector responses, but this

review focuses primarily on Tlymphocytes. There is goodevidence that both CD4+ andCD8+ T lymphocytes play amajor role in determiningoutcome after acute infectionand therefore in the long term.This comes from the followingobservations.

Clearance of acute infection in both man and in chimpanzee models is accompanied bystrong CD4+ and CD8+ T cellresponses against numerous HCVderived antigens. The evidence wasobtained first for CD4+ T cellresponses and initially some specificepitopes were highlighted aspotentially protective. Although

these do appear to be targeted, this isnot exclusive and responses to othergene products are also seen. The

strength of the CD8+

T cell responseagainst one epitope when measuredusing a tetramer, may be up to 8% ofthe total CD8+ T cells, and can includeresponses to at least 8 separateepitopes. By ELISpot analysis, theCD4+ T cell responses appear to be ofa similar magnitude.

Figure: Interactions of HCV proteins withdifferent effectors of the immune response.

The effects of HCV proteins on the

different components of the innate and

specific immunity are summarized.


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The timing of these responses appears to correlate with resolution of viraemia in thosecases where virus is cleared. The level of activation of HCV-specific T cell responses(assessed byCD38expression)

correlates withthe degree ofliverinflammationanalysed byblood ALTlevels.

There is anassociationbetweenpossession of

specific HLAgenes(DRB1*1101and/or DQ1*0301) and spontaneous clearance of virus. This strongly suggests thatselection of particular epitopes is associated with better initial control of viraemia. Thosebearing HLA DQ1*0301 (which is in tight linkage disequilibrium with DRB1*1101)were found to be more likely to possess significant HCV-specific CD4+ T cell responses,further evidence that the responses in these individuals are more robust.

So much for successful responseswhich are in fact the exception. The mechanism for viralpersistence, i.e. failure of Tcell responses in the majorityof patients, is not yet clear.Studies of those who go on todevelop persistent infectionhave highlighted the weakCD4+ T cell responses,although it is not clear yetwhether persistence of viruscauses attenuation of T cellresponses or vice versa. Re-emergence of CD4+ T cellresponses upon clearance ofvirus with interferon-alpha/ribavirin therapysuggests the latter , i.e.suppression of T cells byvirus may be important.


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The picture with regards to CD8+ T cell responses is even less clear. CD8+ T cell responseshave been observed in the acute phase of infection in those who fail to clear virus at levels of 13% of CD8+ lymphocytes against 12 separate T cell epitopes. Whether these are the mainepitopes targeted in these individuals and how, overall, the responses differ in magnitude

between clearers and nonclearers is not known. It appears that failure to clear virus is not due tofailure to mount any CTL response whatsoever, although, like CD4+ T cell responses, these maybe poorly maintained in the face of ongoing viraemia. The overall quality of the response maydiffer in terms of magnitude or breadthor, importantly, peptide selection. It is this latter issuethat forms the focus of this review.Clinical features of HCV

After inoculation, unlike Hepatitis A and B, the acute illness caused by HCV is not welldocumented. This is partly because it is genuinely milder, and possibly it is poorly recognized byphysicians, and also those in the current western risk groups may not present to hospital. This isunfortunate, as it now appears that early intervention is of benefit. When it has been documented,or in animal models, the following features are apparent:

The peak of viraemia may take several weeks to arise. The liver inflammation/liverenzyme level in the blood (usually measured as ALT or AST) does not parallel the viralloadconsistent with the idea that at this stage much of the liver damage is not causeddirectly by the virus.

Resolution of the viraemia(accompanied by cellular immuneresponses) is associated with liverinflammation and in some but not allcases, clinical jaundice. The level ofALT may be 5002000 IU/l, compared

with HBV where it may be 5 or 10times higher. After this period, viraemia either

persists or the individual, in about 15%of cases, becomes RNA negative in theblood. There may be a state ofinstability where virus may become

undetectable in blood temporarily andthen reappears.

If viraemia is established, the level ofviraemia does not correlate with progression of disease, unlike HIV. Disease progression

is measured by the development of liver inflammation, as assessed by blood ALT andhistological indices of lymphocytic infiltration and also by creeping fibrosis. The extentof these vary widely between individuals, and apart from a few factors such as alcoholand coinfection (for example with HIV), the basis of this variation is not understood.


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In those where disease has progressed, therapy (in the form of interferon-alpha andribavirin) may lead to long-term clearance of virus from blood, with accompanyingimprovement in liver histology. The effects of the drugs are not entirely understood, andit is likely that in addition to antiviral activity they influence the immune response, bothdirectly, and indirectly through lowering viral load.

Diagnosis and TestingHow and when is HCV diagnosed?

HCV infection is not infrequently diagnosed as a complete surprise and may causeconsiderable upset for patient and those around the patient including doctor.

The disease can have a broad spectrum of presentations such as:

Major symptoms (swelling of legs and abdomen, confusion)and findings (abnormallaboratory tests (blood) or imagines (Ultrasound, CT scan).

HCV can be diagnosed in a completely asymptomatic individual during health or lifeinsurance check

focused test result after someone's history indicated risk factor's (sometimes decadesago!)

Asymptomatic individual may becomes more symptomatic once knowing that they carryHCV virus. This may due to prior denial of symptoms or occur against the background ofincreased anxiety about the diagnosis.

As part of work-up for a variety of symptoms and findings, often - but not necessarily -including abnormal liver tests:

oFatigueo Jaundice (relatively rare)

o Fluid retention in abdomen or legso Skin and joint complaintso Red blood cells in urineo Abnormal looking liver and/or spleen on ultrasound or CTo Liver tumor on imagingo Mental status changes, disturbance of sleeping pattern

Testing for HCV1)HCV antibodies:

All HIV-infected patients should be tested for HCV infection with the HCV antibody test.Patients with risk factors for HCV infection should be retested at regular intervals. In HIV-Infected patients, the HCV antibody test result sometimes is falsely negative; therefore, if HCVinfection is suspected (e.g., because of a history of high-risk behavior, unexplained elevated


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ALT, or evidence of cirrhosis), the HCV RNA should be tested even if the HCV antibody testresult is negative. A false-negative HCV RNA result is very unlikely in chronic infection.

2)HCV RNA:All patients who test positive for HCV antibody should have HCV RNA testing performed.

As noted above, if patients have negative results on HCV antibody tests but persistentlyabnormal transaminases or suspected acute or chronic infection, HCV RNA testing should beperformed.

The definition of chronic HCV infection is the presence of HCV RNA 6 months after theestimated time of infection. If a patient is HCV antibody positive but HCV RNA negative, thepatient has cleared the HCV and does not have chronic HCV infection.

There are quantitative RNA tests and qualitative RNA tests. Although both types of RNAtests are highly sensitive and specific, thequalitative tests can detect lower levels ofviremia than the quantitative tests. Thechoice of RNA test can be important.

The quantitative RNA tests will bereported as a value, with a measured numberof international units per milliliter (IU/mL).Quantitative tests are useful for determiningthe prognosis of HCV treatment and then

monitoring while on HCV treatment.Qualitative RNA tests will be reported as apresent or absent value, but without anumerical value. They are useful for serialtesting during suspected acute infection andfor determining whether spontaneous viralclearance has occurred, a sustainedvirological response has occurred during

treatment, or a relapse has occurred after treatment.

3)Genotyping:The HCV genotype is the strongest predictor of response to HCV treatment and also is a

critical determinant of the dosage and duration of treatment. HCV genotyping should beperformed once for all patients with detectable HCV RNA; it does not need to be repeated.


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4)Alanine aminotransferase:Monitoring of ALT can be useful to assess acute infection, chronic liver inflammation, and

response to HCV treatment. However, ALT does not always correlate with the degree of fibrosisand in addition, ALT can be persistently normal in 25% of HCV patients, including patients with

cirrhosis or advanced liver disease. Small fluctuations in ALT usually are not clinicallysignificant in HCV, though trends can be significant during or following HCV treatment.

Additional tests:Check complete blood cell count with platelet count, albumin, total bilirubin, and

prothrombin time.

Test all patients for hepatitis B (HBsAg, anti-HBsAb, and anti-HBcAb). Patients with anegative HBsAg and negative anti-HBsAb result should be vaccinated against HBV. Test for

hepatitis A virus (HAV) antibodies (total). All patients with a negative HAV antibody resultshould be vaccinated against HAV.

5)ImagingUltrasonography can be performed to screen for cirrhosis or focal hepatic masses. Computed

tomography (CT), magnetic resonance imaging (MRI), and single-photon emission computedtomography (SPECT) are more expensive and generally are reserved for further evaluation ofliver masses detected by ultrasound.

6)Liver biopsyLiver biopsy is used to define the degree of inflammation (the grade) and degree of fibrosis

(the stage) to determine the need for HCV treatment. Unless there is clear evidence of cirrhosis,laboratory tests and radiology studies are unable to quantitate the degree of fibrosis in the liver.Liver biopsy carries some risk, primarily from bleeding (the risk of significant bleeding orfatality is approximately 1/10,000). Patients with severe thrombocytopenia or coagulopathyshould not undergo liver biopsy. Fibrosis is scored from 0 to 4, with 0 indicating no fibrosis and4 indicating cirrhosis.

Biopsy can be useful in making management decisions for some HCV patients, for example

when determining whether to treat a patient, particularly those with genotype 1 virus (see below).If the biopsy reveals only mild-to-moderate fibrosis, it may be preferable to defer treatment andmonitor the patient. Conversely, if the biopsy reveals more advanced fibrosis, treatment shouldbe considered more urgently. With genotype 2 or 3 patients, some providers consider biopsy tobe unnecessary because treatment outcomes are sufficiently high that findings from a biopsywould not necessarily change the management strategy. For HIV/HCV-coinfected patients, abiopsy may be particularly useful in determining the stage of disease and in planning whether orwhen to initiate HCV treatment, as the course of liver disease may accelerate. Overall, deciding


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whether to conduct a biopsy largely is a matter of individual choice. It is not a requirement fortreatment of any patient, but may be useful for helping the provider and patient make a decisionabout whether or when to undergo treatment.

Test results to detect, diagnose, and monitor HCV include:

Anti-HCV HCV RIBA HCV RNA,Qualitative

HCV Infection

Negative No infection or, rarely,insufficient antibody

Positive Negative No infection; likely a falsepositive

Positive Need to do Negative Likely no infection, pastinfection, or HCV viral loadlow

Positive Positive Negative Past infection or HCV viral load

lowPositive or Weak orIndeterminate

Not done orPositive

Positive Current infection

Indeterminate orPositive

Indeterminate Negative No infection, past infection, orHCV viral load low

Basics of diagnostic tests1.HCV ELISA

An enzyme-linked immunosorbent assay (ELISA) was developed by using a syntheticpolypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus(HCV). Since the introduction in1990 of HCV antibody screening ofblood donations, the incidence of thisinfection in transfusion recipients hasbeen significantly reduced. The firstgeneration of HCV ELISAs showed

limited sensitivity and specificity andwas produced using recombinantproteins complementary to the NS4region of the HCV genome.

Second generation tests, whichincluded recombinant/synthetic


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antigens from the Core and nonstructural regions NS3 and NS4 resulted in a remarkedimprovement in sensitivity and specificity. Clinical studies show that significant amount of HCVinfected individuals develop antibodies to NS5 non-structural protein of the virus. For this, thethird generation tests include antigens from the NS5 region of the viral genome in addition toNS3, NS4 and the Core. The third generation tests have improved sensitivity and have shorten

the antibody detection window period to 70 days.

Principle:This anti-HCV employs solid phase, indirect ELISA method for detection of antibodies

to HCV in two step incubation procedure. Polystyrene microwell strips are pre-coated withrecombinant, highly immunoreactive antigens corresponding to the core and the non-structuralregions of HCV (third generation HCV ELISA). During the first incubation step, anti-HCVspecific antibodies, if present, will be bound to the solid phase pre-coated HCV antigens. Thewells are washed to remove unbound serum proteins, and rabbit anti-human IgG antibodies (anti-

IgG) conjugated to the enzyme horseradish peroxidase (HRP-Conjugate) are added. During thesecond incubation step, these HRP-conjugated antibodies will be bound to any antigen-antibody(IgG) complexes previously formed and the unbound HRP-conjugate is then removed bywashing. Chromogen solutions containing Tetramethylbenzidine (TMB) and urea peroxide areadded to the wells and in presence of the antigen-antibody-anti-IgG (HRP) immunocomplex, thecolorless Chromogens are hydrolyzed by the bound HRP conjugate to a blue-colored product.The blue color turns yellow after stopping the reaction with sulfuric acid. The amount of colorintensity can be measured and it is proportional to the amount of antibody captured in the wells,and to the amount of antibody in the sample respectively. Wells containing samples negative foranti-HCV remain colorless.

Ag(p) + Ab(s) [Ag(p)Ab(s) + ENZ] [Ag(p)Ab(s)ENZ] blue yellow ( + )

Ag(p)+ [Ag(p) +ENZ] [Ag(p) ] no color ( - )

Incubation1 Incubation 2 Immobilized Complex Coloring results

30min 30min 15min

Ag(p)pre-coated HCV antigens(core, NS3/4,NS5);

Ab(s)HCV antibodies in sample (IgG);

ENZHRP conjugated rabbit anti-human IgG.


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2.HCV PCRHCV RNA is first separated from the sample. There are different methods available for doing

so. When viral RNA is separated, its cDNA is formed by a process called Reverse Transcription(RT) by a reverse transcriptase enzyme. Then this DNA fragment is amplified by PCR.

The reverse transcription (RT) and polymerase chain reaction (PCR) amplification stepsare performed sequentially in a single tube. First, genomic HCV RNA is reverse transcribed intocomplementary DNA (cDNA) using HCV-specific primers. Next, the mixture is heated to

activate the DNApolymerase forthe PCRamplification step

and

simultaneouslyinactivate the

reversetranscriptase.

Portions of the 5'UTR and coreregions of theHCV genome are

co-amplifiedfrom the cDNA

using two pairs of biotinylated primers to produce two distinct biotinylated DNA fragments of

240 and 270 base pairs, representing the 5' UTR and core HCV regions respectively. Thenucleotide sequence of the primers has been optimized to yield comparable amplification of sixHCV genotypes. PCR can be used for qualitative as well as quantitative study of HCV.

The Future HCV will continue to put a tremendous burden on future health care expenditures. Many patient have already often irreversible damage. An enormous number of infected patients, many of them as yet unidentified and who

contracted blood through blood products or past iv drug abuse are at risk of developing

cirrhosis with all its complications including hepatocelluar carcinoma. Once advanced cirrhosis with complications, viral eradication (still) very difficult, often

impossible or associated with detrimental complications. Continued contamination / infection through needle transmission & illicit drug use. A fraction of all patients will be eligible for liver transplantation: this is beyond the reach

of most worldwide and if available: the organ shortage is tremendous.

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Finalizing HCV