Hepatitis C Virus

The 1970s saw the emergence of transfusion associated hepatitis which could neither be classified as Hepatitis A or Hepatitis B. Further studies carried out in chimpanzees confirmed the presence of a small lipid enveloped RNA virus which was transmitted as a blood-borne pathogen as the causative agent for non-A, non-B hepatitis. The late 1980s saw the joint discovery by Michael Houghton, Chiron Diagnostics, Daniel Bradley, Centers for Disease Control, USA, of the virally encoded antigen of this non A-non B hepatic C Virus. Soon after, the complete viral genome was encoded .This new agent named Hepatitis C was associated with the establishment of persistent infection and had a strong association with liver cirrhosis and carcinoma.

Classification

Hepatitis C has been classified in the family Flaviviridae under a separate genus Hepacivirus.  HCV is an enveloped virus with a positive sense, single stranded RNA constituting the genome of the virus. The genome codes for many structural and nonstructural proteins. The three main structural proteins are:

  • the nucleocapsid, Core (C)
  • the two envelope proteins
    1. E1
    2. E2
  • the nonstructural proteins (NS1 to NS5) NS3, NS4A, NS4B, NS5A, NS5B which are involved in viral replication.

The viral replication takes place in the hepatocytes and the peripheral mononuclear cells originating from the lymphoid organs. The half life of the virions in the blood stream is 2.5 hours and up to 1.0 X 1012   virions are produced each day in a chronically infected patient. The high level of turnover seen in the virus leads to rapid accumulation of mutations. At any given time, multiple variants of HCV can be recovered from the plasma and the hepatocytes of the same infected individual. Hence a quasi-species or ?swarm? of closely related but genetically distinct clones of HCV exists in each infected individual. This is also known as quasi-species variation.

HCV genotypes

HCV also shows marked heterogeneity according to geographical regions. There are a minimum of six major genotypes of HCV .Each individual genotype can be further divided into subtypes which share 75% to 85% of the nucleotide sequence identity with the core -E1 and the NSB5 regions.

Clinical features

  • Acute hepatitis C: the average incubation period is approximately 7 weeks which falls in between that of Hepatitis A and Hepatitis B. The patient presents with nonspecific symptoms such as nausea, vomiting, malaise and dark colored urine. The specific liver enzymes are elevated, though not as much as in Hepatitis A or B. Prospective studies of transfusion induced hepatitis have shown that as many as 75% of these patients can be anicteric.
  • Fulminant Hepatitis C:  This is an uncommon presentation in the western population but is more commonly seen in Japan for reasons yet unexplained. About 40% to 60% of fulminant hepatitis of non-A, non B origin are attributed to Hepatitis C.
  • Chronic Hepatitis C: HCV is a known causative agent of chronic liver disease. Approximately 50% to 85% of individuals with acute infection develop persistent disease. Chronic HCV infection persists for many decades negatively affecting the quality of life of the patient. Fibrosis around the portal triads, which eventually progresses to cirrhosis of the liver, is a common feature of chronically infected patients. Of these 5 % to 20% decompensate within 5 years with the development of the following:
    1. esophageal varices
    2. coagulopathy
    3. ascitis
    4. encephalopathy
    5. hepatocellular carcinoma.
  • Extrahepatic manifestation of hepatitis C: HCV infection is strongly associated with essential mixed cryoglobulinemia, membranoproliferative glomerulonephritis and porphyria cutanea tarda. Approximately one-half the HCV infected patients have circulating cryoglobulins. About 60 % to 80 % patients of sporadic porphyria cutanea tarda are found to be HCV infected. Women with chronic HCV infection have been found to be affected with autoimmune thyroid disorders such as Hashimoto?s thyroiditis.

Laboratory diagnosis of HCV infection

  • Serology: Enzyme immunoassays (EIA) are the most common methods used for detection of HCV infection. The second generation assays previously utilized detected antibodies to the NS4, core and NS3 sequences. Third generations assays with 97% sensitivity have additional antibody detection to NS5, core (reconfigured) and NS3 antigens. The third generation EIAs are capable of detecting infections within 6 to 8 weeks of exposure. The confirmatory assay as approved by the US FDA is the Recombinant Immunoblot assays (RIBA). RIBA identifies two specific antigens to which the EIA has reacted. Third generation RIBA has the maximum sensitivity and therefore reduces the number of indeterminate results.
  • Viral RNA detection: The viral RNA can be detected by molecular methods such as polymerase chain reaction (PCR), real time PCR, transcription mediated amplification or branched DNA technologies. Molecular methods assist in the quantification of the viral loads which is especially important in assessing the response to treatment in patients who are undergoing antiviral therapy. It has been determined that numerous factors affect the estimation of HCV RNA in the serum or plasma such as the assay used, storage temperature, type of vial used for blood collection and the testing laboratory. Therefore, it is better to quantify the results in International Units (IU) which correspond to a standardized value of HCV RNA than the number of viral particles.
  • Antigen detection: The HCV core protein can now be detected reliably using chemiluminescence assay developed by Ortho-Clinical Diagnostics, Raritan, N.J. This technique is comparable to the viral RNA detection and is technologically less demanding than the enzyme immunoassay methods.
  • Genotyping: determining the viral genotype assists in predicting the response to therapy. Genotyping is done by sequencing the viral genome (E1 , NS5 regions).Three assays are available for genotyping 1. Trugene HCV 5? NC (Visible Genetics, Toronto, Ontario, Canada) 2. Line Probe Assay INNO- LIPA HCV II (Innogenetics Ghent, Belgium) based on reverse dot ? blot hybridization of PCR amplicons to nitrocellulose paper strips coated with specific genotypic probes. This assay is known to accurately identify the genotype of the virus which is required for clinical decision making, though it may sometimes misclassify the subtype of the virus. 3. Chiron Corporation (Emeryville, California) has developed a serologic method to establish the genotype of the virus. This method is incapable of classifing the subtype of the virus.

Diagnosis of HCV infection

The preferred method for screening is an Enzyme Immunoassay or a Chemiluminescence assay. The reactive sera can then be subjected to molecular methods for quantification and genotyping. A negative RNA test with a positive HCV antibodies test indicates that the infection has resolved.

The RIBA is a useful test in determining whether the infection has resolved or or is persisting.  This is determined by discrepant results obtained between the EIA and HCV RNA detection. A EIA positive, HCV RNA negative or RIBA negative indicates a false positive EIA results thus requiring no further testing of the individual, particularly when there are no risk factors such as when persons are tested for insurance. In the event of the presence of risk factors, a repeat HCV RNA and serum Alanine aminotranferase test (ALT) must be done after six months. If the HCV RNA is negative and the ALT concentration is also normal, it indicates either a resolution of infection or a false positive EIA.

In the event of strong clinical suspicion  such as in acute liver disease or immunocompromised patients having a negative result for an EIA, it is expedient to do HCV RNA which can easily be localized up 14 days after exposure. Antibodies to HCV take at least 8 weeks to be detected in the serum after exposure. HCV RNA estimation is also a useful test in patients who have a diminished antibody response such as HIV positive individuals, patients in chronic renal failure who are dependent on hemodialysis and those with congenital immunodeficiency syndromes.