MOLECULAR ASSAYS

The demonstration of hepatitis C viral particles in blood confirms the diagnosis of hepatitis C infection. Two principal methods used to detect hepatitis C viral RNA are target amplification and signal amplification.

Target amplification assays such as the polymerase chain reaction (PCR) rely on sequence-specific primers and a heat-stable DNA polymerase to generate a large number of copies of a portion of the viral genome. Signal amplification-as used in DNA assays, uses a series of hybridization reactions between probes specific for several regions of the target molecule and subsequent hybridization to a DNA amplifier.

Viral load may be measured as either a qualitative or quantitative function. Qualitative testing is the most sensitive and specific and, therefore, the most accurate when used for initial diagnosis. Both qualitative and quantitative viral load testing have a role in the evaluation and treatment of patients with hepatitis C. Qualitative testing is important in confirming a positive anti-HCV test and in assessing sustained response to therapy. Quantitative testing is useful in determining diagnosis, predicting response to therapy, and monitoring response while on therapy.

Many different “brands” of quantitative testing are available and until recently, results could not be compared across assays because of a lack of standardization. As of the year 2000, results of all quantitative assays are standardized as international units per milliliter. Viral load testing is useful in evaluating the patient with suspected acute hepatitis C infection because these assays should be positive within 1-2 weeks of initial exposure. These tests, however, do not correlate with disease severity or the rate of disease progression.

Hepatitis C core antigen immunoassays, currently undergoing testing, may approach the clinical sensitivity of HCV-RNA testing.

Genotype:

Genetic analysis of HCV reveals the existence of numerous viral sequences, termed genotypes. These various genotypes differ in genetic composition by as much as 35%. Six major genotypes have been identified and these can be further subdivided into more than 100 subtypes.

Genotype distribution is worldwide. However, 1a and lb are the most common types in the United States, accounting for more than 75% of all infections. Genotype lb is the most prevalent viral species found in Japan. Genotype 3, which is uncommon in the United States except in younger intravenous drug users, is, however, highly prevalent on the Indian subcontinent. Genotype 4 accounts for the majority of HCV infection in Egypt and is also seen in other areas of Africa. Genotype 5 is common in South Africa and accounts for more than 50% of all cases of hepatitis C seen in that region. Finally, genotype 6 is found primarily in Southeast Asia.

Hepatitis C genotype does not appear to affect the rate of disease progression. Genotype is, however, a predictor of response to therapy. Patients with genotype 2 or 3 are more likely to respond to therapy and based on published data, may be treated with combination interferon and ribavirin for a 6-month course. By contrast, patients infected with HCV genotypes 1 and 4 are less likely to respond to therapy and should be treated with combination interferon and ribavirin therapy for 1 year. Genotyping should be performed in all patients with hepatitis C for whom treatment is being considered. Obtaining the results of genotype testing can allow a more cost-effective approach to therapy.

Liver Biopsy:

The grading and staging of liver disease in patients with hepatitis C is crucial in helping guide treatment. Unfortunately, serum aminotransferase levels, HCV viral load, and hepatitis C genotype are all poor predictors of underlying histology. Therefore, despite the cost and discomfort associated with liver biopsy, it is recommended that it be performed in the initial evaluation of all hepatitis C patients, barring obvious contraindications. Information obtained from the biopsy can help exclude other causes of liver disease, help gauge the rate of progression of disease, and aid the treating physician in adjusting or terminating therapies if the patient is experiencing significant side effects.