Serologic Tests for Chronic Hepatitis C

Enzyme Immunoassay

Anti-HCV is detected by enzyme immunoassay (EIA). The third-generation test (EIA-3) used today is more sensitive and specific than previous ones. However, as with all enzyme immunoassays, false-positive results are occasionally a problem with the EIA-3. Additional or confirmatory testing is often helpful.

The best approach to confirm the diagnosis of hepatitis C is to test for HCV RNA using a sensitive assay such as polymerase chain reaction (PCR) or transcription mediated amplification (TMA). The presence of HCV RNA in serum indicates an active infection.

Testing for HCV RNA is also helpful in patients in whom EIA tests for anti-HCV are unreliable. For instance, immunocompromised patients may test negative for anti-HCV despite having HCV infection because they may not produce enough antibodies for detection with EIA. Likewise, patients with acute hepatitis may test negative for anti-HCV when first tested. Antibody is present in almost all patients by 1 month after onset of acute illness; thus, patients with acute hepatitis who initially test negative may need followup testing. In these situations, HCV RNA is usually present and confirms the diagnosis.

Recombinant Immunoblot Assay

Immunoblot assays can be used to confirm anti-HCV reactivity as well. These tests are also called "Western blots"; serum is incubated on nitrocellulose strips on which four recombinant viral proteins are blotted. Color changes indicate that antibodies are adhering to the proteins. An immunoblot is considered positive if two or more proteins react and is considered indeterminate if only one positive band is detected. In some clinical situations, confirmatory testing by immunoblotting is helpful, such as for the person with anti-HCV detected by EIA who tests negative for HCV RNA. The EIA anti-HCV reactivity could represent a false-positive reaction, recovery from hepatitis C, or continued virus infection with levels of virus too low to be detected (the last occurs only rarely when sensitive PCR or TMA assays are used). If the immunoblot test for anti-HCV is positive, the patient has most likely recovered from hepatitis C and has persistent antibody. If the immunoblot test is negative, the EIA result was probably a false positive.

Immunoblot tests are routine in blood banks when an anti-HCV-positive sample is found by EIA. Immunoblot assays are highly specific and valuable in verifying anti-HCV reactivity. Indeterminate tests require further followup testing, including attempts to confirm the specificity by repeat testing for HCV RNA.

Direct Assays for HCV RNA

PCR and TMA amplification can detect low levels of HCV RNA in serum. Testing for HCV RNA is a reliable way of demonstrating that hepatitis C infection is present and is the most specific test for infection. Testing for HCV RNA is particularly useful when aminotransferases are normal or only slightly elevated, when anti-HCV is not present, or when several causes of liver disease are possible. This method also helps diagnose hepatitis C in people who are immunosuppressed, have recently had an organ transplant, or have chronic renal failure. A PCR assay has now been approved by the Food and Drug Administration for general use. This assay will detect HCV RNA in serum down to a lower limit of 50 to 100 copies per milliliter (mL) which is equivalent to 25 to 50 international units (IU). A slightly more sensitive TMA test is currently under evaluation and may soon become available. Almost all patients with chronic hepatitis C will test positive by these assays.

Quantification of HCV RNA in Serum

Several methods are available for measuring the concentration or level of virus in serum, which is an indirect assessment of viral load. These methods include a quantitative PCR and a branched DNA (bDNA) test. Unfortunately, these assays are not well standardized, and different methods from different laboratories can provide different results on the same specimen. In addition, serum levels of HCV RNA can vary spontaneously by 3- to 10-fold over time. Nevertheless, when performed carefully, quantitative assays provide important insights into the nature of hepatitis C. Most patients with chronic hepatitis C have levels of HCV RNA (viral load) between 100,000 (105) and 10,000,000 (107) copies per mL. Expressed as IU, these averages are 50,000 to 5 million IU.

Viral levels as measured by HCV RNA do not correlate with the severity of the hepatitis or with a poor prognosis (as in HIV infection); but viral load does correlate with the likelihood of a response to antiviral therapy. Rates of response to a course of alpha interferon and ribavirin are higher in patients with low levels of HCV RNA. There are several definitions of a "low level" of HCV RNA, but the usual definition is below 1 million IU (2 million copies) per mL.

In addition, monitoring HCV RNA levels during the early phases of treatment may provide early information on the likelihood of a response. Yet because of the shortcomings of the current assays for HCV RNA level, these tests are not always reliable guides to therapy.

Genotyping and Serotyping of HCV

There are 6 known genotypes and more than 50 subtypes of hepatitis C. The genotype of infection is helpful in defining the epidemiology of hepatitis C. More important, knowing the genotype or serotype (genotype-specific antibodies) of HCV is helpful in making recommendations and counseling regarding therapy. Patients with genotypes 2 and 3 are two to three times more likely to respond to interferon-based therapy than patients with genotype 1. Furthermore, when using combination therapy, the recommended dose and duration of treatment depend on the genotype. For patients with genotypes 2 and 3, a 24-week course of combination treatment using interferon and 800 milligrams (mg) of ribavirin daily is adequate, whereas for patients with genotype 1, a 48-week course and full dose of ribavirin (1,000 to 1,200 mg daily) is recommended. For these reasons, testing for HCV genotype is often clinically helpful. Once the genotype is identified, it need not be tested again; genotypes do not change during the course of infection.

Biochemical Indicators of Hepatitis C Virus Infection In chronic hepatitis C, increases in the alanine and aspartate aminotransferases range from 0 to 20 times (but usually less than 5 times) the upper limit of normal. Alanine aminotransferase (ALT) levels are usually higher than aspartate aminotransferase (AST) levels, but that finding may be reversed in patients who have cirrhosis. Alkaline phosphatase and gamma glutamyl transpeptidase are usually normal. If elevated, they may indicate cirrhosis. Rheumatoid factor and low platelet and white blood cell counts are frequent in patients with severe fibrosis or cirrhosis, providing clues to the presence of advanced disease. The enzymes lactate dehydrogenase and creatine kinase are usually normal. Albumin levels and prothrombin time are normal until late-stage disease. Iron and ferritin levels may be slightly elevated.

Normal Serum ALT Levels

Some patients with chronic hepatitis C have normal serum alanine aminotransferase (ALT) levels, even when tested on multiple occasions. In this and other situations in which the diagnosis of chronic hepatitis C may be questioned, the diagnosis should be confirmed by testing for HCV RNA. The presence of HCV RNA indicates that the patient has ongoing viral infection despite normal ALT levels.

source: http://digestive.niddk.nih.gov/ddiseases/pubs/chronichepc/index.htm