Indian Journal of Medical Sciences Medknow Publications on behalf of Indian Journal of Medical Sciences Trust ISSN: 0019-5359 EISSN: 1998-3654 Vol. 57, Num. 10, 2003, pp. 461-468

Indian Journal of Medical Sciences, Volume 57, Number 10, October 2003, pp. 461-468

Practitioners section

VIRAL HEPATITIS (Part - III)

D D Banker*

Correspondence: D. D. Banker Consultant Pathologist, Mumbai, India.

Accepted Date: 01-07-2003

Code Number: ms03033

HEPATITIS C (HC)
Nearly 170 million people worldwide have been infected with HCV. About 85% of these develop persistent infection and are at risk of long term complications like liver cirrhosis and HCC. Hepatitis C (HC) was a part of non A - non B (NANB) group of hepatitis for many years after identification of hepatitis B. In 1989 the genome of hepatitis C virus (HCV) was identified as a separate entity by Choo, et al in USA, using a recombinant complimentary DNA (cDNA) approach.^1,2

At least 50 % of the patients previously designated as post-transfusion hepatitis (PTH) cases due to NANB are now known to be that of HCV infection. With the elimination of HBsAg positive blood, HCV has now replaced HBV as the commonest cause of PTH in many countries. A large number of HC infections also occur among injection drug abusers, organ transplant cases, hemodialysis patients and those having multiple transfusions such as thalassemia and hemophilia cases. Vertical transmission from mother to infant may also occur. Approximately half of HCV patients develop chronic active hepatitis and this may progress to liver cirrhosis and hepatocellular carcinoma (HCC). Serological tests (ELISA and PCR) have been developed for the diagnosis of the infection. Immunity after HC is short-lived or non-existent and active immunization is not possible at present. Immunization against HCV is not yet available as one HCV envelope protein undergoes constant evolution, thus defying vaccine development. Normal human immunoglobulin (Ig) may provide some passive protection against postexposure infection.

HC has become an increasingly important public health problem in recent years.³ Although treatment of HCV with interferon alpha-2 alone or in combination with ribavirin helps in delaying disease progression, the treatment involves a long period of injections and the use of drugs that produce many side effects. Prevention of spread requires identification of patients with HCV as the most common blood-borne chronic infection. Chronic HC accounts for 40 % of the deaths from chronic liver disease. In over 20% of those with chronic HC cirrhosis will develop in about 20 years. The development of cirrhosis worsens prognosis and leads to higher risk of HCC, liver transplantation where facilities exist, end-stage liver decompensation and death. Medical and work loss cost of this disease is millions of rupees. Recombinant interferon alpha-2a for a 12 month course is the approved treatment. Interferon has two effects on HCV-it directly kills the virus and it helps the body's own defence against the virus. Combining ribavirin with interferon had shown some advantage.

CLINICAL FEATURES
The clinical course of untreated HCV infection is highly variable with the majority of patients experiencing a slow fluctuating disease that may take 20 years or more for full expression. The incubation period is about 7 weeks with a range of 2 to 26 weeks. After initial exposure HCV RNA can be detected in serum by PCR within a few weeks. Most patients develop liver cell injury within a few months as indicated by elevated ALT. A large percentage of patients are physically asymptomatic and anicteric. A "silent period" may ensue for weeks to month after initial infection, during which viral titers are low and antibody responses are not detectable. Seroconversion to HCV antibody positive status occurs within 3 months in the majority of the exposed patients but in some it may take upto 6 months. Fulminant hepatitis C is extremely rare. HCV is not efficiently cleared by the immune system after acute infection and about 80% progress to chronic infection. Approximately 25% of chronically infected patients will progress to cirrhosis. The estimated interval from infection to development of chronic hepatitis is about 14 years, to chronic active hepatitis 18 years, for cirrhosis 21 years and for HCC 28 years. Factors which may affect the rate of progression are age, sex and amount of alcohol consumption. As with chronic HB, chronic HC is also associated with increased risk of HCC.

Chronic HC is defined as the presence of HC for longer than 6 months. Chronic HCV infection follows an insidious course involving episodes of elevated serum aminotransferases and hepatocellular injury, often associated with fluctuations or increases in HCV titer. Nonspecific physical symptoms such as weight loss, fatigue, muscle or joint pains, irritability, nausea, malaise, anorexia and pain in hepatic region develop in about 20 % of the cases. Considerable morbidity occurs in chronic HC and the development of severe fibrosis and necro-inflammatory changes in liver shows poor prognosis suggesting progression to cirrhosis. Cirrhosis leads to liver failure in some of the cases.

EPIDEMIOLOGICAL FEATURES AND TRANSMISSION
HCV infection is present worldwide and the varying prevalence of the disease depends on the geographical area. The highest prevalence of HC is found among 30-49 age group. HCV is a blood-borne virus that usually circulates in low titres in infected serum.

Transmission of HCV is primarily by parenteral route like HBV. The incidence is reduced in developed countries where blood banks are employing tests for presence of HCV antibodies or RNA before collection of blood but so far this is woefully neglected in many developing countries including India.⁴ HCV testing of suspected asymptomatic persons or items should be carried out in injection drug abusers either current or former, blood or blood product, recipients of untested blood or products, hemodialysis patients, persons with persistently elevated alanine aminotransferase concentration, blood donors, organ donors, children born to HCV positive women and healthcare workers with percuntaneous or mucosal exposure to HCV positive blood.

Epidemiologic studies have shown that besides use of blood transfusion, blood products and transplantation of organs from infected donors, the most common modes of transmission are the sharing of contaminated needles by injection drug users. The effective screening of blood donations by intensive questionnaire and the use of specific 3rd generation HCV antibody testing can greatly reduce the risk of transmission from blood to negligible levels. PCR-RNA test would pick up any possible infection during the early months of infection (window period). Pooled plasma or its derivatives have a high risk of transmission e.g. clotting factors used for hemophiliacs. Hemodialysis patients have a seropositivity rate of 10 % to 40%. Sexual exposure accounts for 10% to 20% of new cases, but this is most prevalent among persons who engage in high risk sexual practices such as not using condoms or having multiple partners. The risk of HCV infection from an HCV-positive partner in a long term monogamous relationship is as low as 1.5%. The risk of HCV transmission after a single exposure is negligible. The other risk factors besides hemodialysis are occupational, household and perinatal exposures which combined account for 10% of all HCV infections. Viral transmission to infants from HCV infected mothers is approximately 5%. The rate increases when the mother is coinfected with HIV. Breast feeding is not important in transmission of HCV, hence it is not contraindicated. Because of the high prevalence of this disease, transmission prevention counseling is an important step in disease management. Of the 170 million people worldwide who have evidence of HCV infection, about 66% are chronically infected. Chronic HCV infection leads to 8,000 to 10,000 deaths annually. The frequency of patients with complications of HC is expected to triple within the next 20 years, resulting in a large increase in incidence of cirrhosis and HCC, and an increased demand for liver transplantation. This will throw a heavy burden on treatment for the disease all over the world. HCV is now known to be responsible for most of the transfusion associated hepatitis formerly called non A - non B. Perinatal transmission is approximately 5% with a very low risk of chronic infection developing in the infant.

The global prevalence of HCV carriers is estimated to average 3% ranging from 0.1 to 10% or more in different countries. In northern Europe, it is about 0.5%, in southern Europe 2% and in eastern Europe 0.7 to 5%. There is high prevalence in Mongolia, Vietnam, Myanmar,China, Central Africa and Egypt. In north America prevalence is low at about 1%. In south America there is high prevalence in Brazil. The highest prevalence of 10% or more is seen in Mongolia, Egypt, Tanzania, Ceisinea and Cameroon. Spontaneous disappearance of HCV in patients with chronic HCV liver disease is rate. Chronic HC is often associated with extrahepatic manifestation such as cryoglobulinemia. Although mild chronic inflammation is present in most cases, progressive liver disease leading to cirrhosis occurs in approximately 25% of patients over 2 to 3 decades and in about 30 years HCC develops.

NATURAL HISTORY
Because of its capacity for rapid mutation and more than 80 subtypes, HCV has the ability to escape host immune attack and this has rendered vaccine development very difficult. The average incubation period after exposure to HCV is about 7 weeks. However HCV RNA appears in serum within 1-3 weeks after exposure whereas anti-HCV antibody can be detected only after about 5 or 6 months after the onset of inspection. Symptoms including malaise, weakness, anorexia arthralgia, low grade fever and icterus ate usually very mild or subclinical and develop only in one-third or less of exposed persons. Serum ALT levels also are not diagnostic. Chronic infection occurs in 85% if clinical symptoms do not resolve within a few months. Fulminant hepatitis is rare. HCV can establish chronic infection even though there may be active humoral and cellular immune responses in the host. Although acute infection with HCV is relatively benign, chronic infection can cause serious liver injury. Clinical characteristics of the symptoms caused in acute hepatitis due to HA. HB or HC are indistinguishable, except that acute HCV infection is more often subclinical.

Many HCV patients are not identified because parameters such as antibody and ALT levels are variable. The course of chronic HC is insidious and most infected persons remain asymptomatic until an advanced stage of disease. Within 20 years of infection cirrhosis will develop in at least 15 to 20% of patients and HCC will develop in 1 to 5%. Within 10 years of the development of cirrhosis, as many as 20% of patients will either exhibit HCC, require transplantation or die of the HCV-related liver disease. The severity of the inflammation is a major factor in the progression of HC to cirrhosis. This has prompted treatment efforts to reduce inflammation and slow down disease progression. Liver disease secondary to HCV infection is estimated to cause death in approximately 3% of patients.

Since the histopathologic features of HCV are nonspecific, liver biopsy is not important in establishing the diagnosis of HC. On the other hand, establishing the presence of cirrhosis or stage of liver disease has been an important prognostic factor not only for development of HCC but in predicting the future course of chronic hepatitis C, whether or not fibrosis or cirrhosis is present.

Factors that may hasten progress of liver disease include alcohol consumption, viral characteristics (e.g. genotype 1, high viral load), male gender, older age at first exposure and coinfection with other viruses such as HB or HIV. Liver failure is a leading cause of death in many parts of the world.

The natural history of HCV in childhood is not well understood. The clinical picture of acute hepatitis in children is same as for HA and HB, Blood transfusion e.g. for thalassemia or hemophilia seems to be the principal route of HCV infection in children. Perinatal transmission is rare. HC in children is uncommon, the incidence being 0.2 to 0.4%. Diagnosis of perinatal transmission relies on HCV-RNA testing. Maternal anti-HCV may persist until 18 months of life giving false positive results. Infants more than 18 months of age and children suspected of HC should be periodically tested for anti HCV.

The presence of HCV antibody by 2nd or 3rd generation ELISA strongly suggests HCV infection, especially in patients with a risk factor for acquiring HCV. In lmmunosuppressed patients antibody test may not be useful and HCV RNA test by PCR should be conducted in them. Among risk factors are: religious scarification, intravenous drug use, intranasal cocaine use, exposure to needles, sharp instruments, blood or blood products, tattooing, immunoglobulin injections, treatment for schistosomiasis, sexually transmitted disease, having had more than 50 sexual partners, male homosexual or infection with HIV or HBV. Patients with a risk factor should undergo testing for HBV, HCV, and HIV.

SEROLOGY
Screening tests for HC include ELISA for anti-HCV and HCV-RNA by PCR.⁵ Acute HC cannot be reliably diagnosed by antibody test as these often do not give positive results upto three months after exposure (window period). HC is still the cause of very large number of post-transfusion hepatitis.

Since the HCV genome was cloned in 1989 (ref), the extensive use of molecular techniques has led to the development of serologic and molecular tests for detecting the virus and its subtypes. It has also added to our understanding of the pathogenesis of chronic hepatitis. Two types of virologic assays are used for the diagnosis and management of HC:

1. Serologic assays based on HCV antigen for HCV antibodies (anti HCV) and
2. Molecular based assays on the quantitation and characterization of HCV RNA.

HCV ANTIBODY TESTS
ELISA has been the major screening test for the detection of anti-HCV against different HCV antigens. A refinement of this is the recombinant immunoblot assay (RIBA). RIBA in rather expensive but is useful in detecting false positive ELISA tests that may be seen in person with no apparent risk factor for HC and those having other immune-mediated diseases such as rheumatoid arthritis. In high risk persons, a positive 2nd or 3rd generation ELISA test is over 95% certain and In the presence of elevated ALT levels establishes the diagnosis.

HCV RNA TESTS
These are useful in:

1. Some patients who may be unable to mount sufficient serologic response,
2. During "window period", and
3. To assess recovery after antiviral treatment, as antibody response generally indicates infection but does not distinguish between present and previous infection. molecular assays include measuring HCV virus load-a parameter that estimates the level of virus replication-, and tests analyzing the sequence of HCV genomes or `genotyping'.⁶ Qualitative tests for detection of HCV RNA in serum, reverse transcription (RT) - PCR or branch DNA (bDNA) methods have been useful in the diagnosis of active HCV infection. Accurate quantitative methods for measuring viral nucleic acid levels, i.e. counting the number of viral particles in patient's serum have also been shown to correlate with the clinical stage of disease. Detection of HCV RNA is definite proof of HCV infection and changes in its titer indicate patient response to antiviral therapy. HCV RNA test has replaced RIBA because the former besides definite diagnosis, also provides information on virus load.

HCV GENOTYPING AND SUBTYPING
HCV has been classified into multiple strains or genotypes on the basis of the identification of their different genomic sequences into genotypes 1 to 6.⁶ The genotypes have been further differentiated into subtypes and quasitypes. Specific HCV genotypes vary in different populations. Genotypes 1 and 2 are common in North America and Europe. Genotype 3 is more common in India. Genotype 4 is more common in West Asia and Egypt. Genotype 1 b is considered to have a more aggressive course of liver disease. The main role of HCV genotyping is to predict the likelihood to maintenance of long term response to therapy, as the genotypes are associated with susceptibility to antiviral therapy.⁷

FURTHER TESTING OF HCV ANTIBODY POSITIVE PATIENTS
PCR testing for HCV RNA is the most sensitive method for the detection of HCV infection and is also used to measure viral load in serum or blood. PCR is the test of choice in patients with normal levels of liver enzymes and no risk factors for HCV. The HCV genotype is useful to predict the chance of "cure" or sustained response, which is conveyed by PCR results that are persistently negative after treatment. Combination therapy with interferon and ribavirin should be used for 12 months in patients with genotype 1 and for 6 months in patients with any other genotype. Among the causes of chronically elevated liver enzymes ALT or AST are obesity, alcohol abuse or dependence, medications that are hepatotoxic, other viral infections, iron overload and other illnesses. Liver biopsy should be considered in patients with signs of progressive liver disease, such as normal liver function test values, low white blood cell count or low platelet count.

PREVENTION
Methods for prevention of HC are similar to those of HB, namely adequate sterilization and disinfection of medical equipment, and environmental surfaces. HCV positive persons must not donate blood, organs, tissues or semen. Among household contacts sharing of toothbrushes and razors must be avoided. Safe sexual practices, including use of latex condoms should be practised by persons having multiple sexual partners. Needle sharing among injection drug users must be avoided. For HCV neither vaccine nor passive immunization is so far available. Recent reports of severe cases of HA occurring in patients with preexisting chronic HC suggest that patients with chromic liver disease who are HAV HBV seronegative should receive prophylactic immunization against these infections.⁸ Similarly HC patients who have undergone liver transplantation should be screened first for HAV and HBV antibodies and given appropriate vaccine if they do not have evidence of immunity.

Sexual partner(s) of HC patients and children born to mothers with chronic HC should be tested for presence of HCV infection even though the risk of sexual or vertical transmission is less than 5%. Patients suffering from HC are likely to suffer additional severe hepatitis due to infection with other hepatitis viruses. Therefore, chronic HC patients should be immunized against HAV and HBV. HC has now been associated with a wide variety of extrahepatic diseases including cryoglobulinemia that may lead to multiorgan injury and death. HC patients with immune complex disease should be given interferon therapy. All patients with chronic HC should be examined for nenal damage by measuring serum creatinine levels and albuminuria levels as well as physical testing to detect neuropathy and vasculitis.

Coinfection with HCV and HBV increases the risk of cirrhosis and decompensated liver disease. HIV coinfection with HCV indicates a poor outcome such as end stage liver disease. Therefore in patients with cirrhosis detected by liver biopsy or with clinical evidence of decompensated liver disease, screening for liver cancer should be carried out with alpha-feto protein level estimation and ultrasonography six-monthly or yearly. Chronic HC occurs in 85% of patients with acute infection and evolves into cirrhosis, HCC and extrahepatic diseases in many patients.

In general practice the following persons should be screened far HCV infections : those who have or might have received blood products before sensitive ELISA were introduced, hemophilia and thalassemic patients, patients undergoing hemodialysis, children born to mothers who have HC and current or previous users of IV drugs. ELISA results may be negative in immunosuppressed patients with chronic HC or in those who have chronic HC and are undergoing hemodialysis. In these patients detection of HCV RNA by PCR should be done.

MOTHER TO INFANT TRANSMISSIONS
Children born to HCV infected mothers usually exhibit HCV antibodies between a few months and one year because of passive transfer from the mother. Hence diagnosis of HCV infection in baby is based on detection of HCV RNA by PCR. When PCR results are negative, the lack of transmission is confirmed by the gradual disappearance of HCV antibodies.⁹

ACCIDENTAL EXPOSURE
In accidental exposure to HCV infected blood, HCV RNA can be detected in serum as early as the second week after exposure. Elevated ALT and seroconversion are observed a few weeks later in the absence of therapy. Antiviral therapy for HCV is not an emergency and may be initiated after the diagnosis of acute HCV infection is made. Antiviral therapy prevents progression to chronic disease in a large number of cases. Prognosis and assessment of severity are best evaluated by means of liver biopsy.

TREATMENT
There is an alarming frequency of HCV infection and persistence among injection drug users. It has been found that immunity against viral persistence can be acquired and vaccines should be tested to reduce the burden of HCV-related liver disease. Although therapy with interferon alpha and ribovarin gives a sustained response rate of 40%, these new therapies are expensive for developing countries where vaccine would be an important alternative way to control this disease. But vaccine is not yet available.

The goal of therapy is to clear HCV RNA from serum. Hence HCV RNA should be measured before starting therapy, after 6 months of combination therapy or 3 months of interferon monotherapy, at the completion of treatment (end of treatment response-ETR) and 6 months after the completion of treatment (i.e. sustained response-SR), Patients receiving combination therapy without viral clearance after 6 months are not likely to respond to further treatment.¹⁰

Early treatment with interferon alpha reduces the risk of chronic infection. The rate of chronicity in untreated patients is about 80% while treatment with interferon reduces this to about 50%.

The decision to treat patients with chronic HC is based on the combination of elevated serum ALT, chronic hepatitis with or without evidence of cirrhosis on liver biopsy and HCV RNA detection by PCR. Qualitative HCV RNA detection assays which are more sensitive than current quantitative assays must be used to assess the virologic response of patients with chronic HC to antiviral therapy. Combination of interferon (IFN) alpha 2b and ribivarin has considerably improved the management of chronic HC.

OUTCOMES OF TREATMENT
Four kinds of virologic response may be obtained:

1. Sustained response(SR) in which HCV RNA assessed by sensitive reverse transcription PCR (RT-PCR) is undetectable in serum or plasma,
2. Virologic relapse in which after undetectable phase the HCV RNA recurs,
3. Virologic breakthrough in which after temporary undetectable phase HCV RNA is again detected during contiunous therapy, and
4. Virologic nonresponse in which there is failure to suppress HCV RNA at any point during therapy. Sustained virologic response correlates with histologic and clinical improvements. Almost 50 % of patients treated with IFN alpha alone experience virologic and/or biochemical relapse. At present there are no approved therapies for the treatment of virologic nonresponders.^11-14

SIDE-EFFECTS OF TREATMENTS
These are mostly minor. Ribivarin may cause dose-related hemolytic anemia that usually occurs during the first four weeks of treatment. Contraindications to administration of IFN alpha include severe depression or other psychiatric disorders, active drug or alcohol abuse and autoimmune disorders such as rheumatoid arthritis or lupus erythematosus.

FUTURE AND EMERGING ANTI-HC THERAPIES

1. Pegylated IFN-alpha in which IFN-alpha is modified by the covalent attachment of polyethylene glycol (PEG) that makes it more effective by prolonged serum half-life allowing once weekly administration,
2. Helicase inhibitors which prevent the unwinding of double-stranded viral RNA during HCV replication,
3. Protease inhibitors which prevent the cleavage of large viral proteins into smaller segments,
4. RNA polymerase inhibitors which prevent replication or copying of HCV genome,
5. Antisense nucleotides which bind to IFN resistance sites,
6. DNA vaccines which stimulate cytotoxic T-cell activity.

REFERENCES

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