Hcv Rna Detection and Quantitation

Detection of HCV RNA in serum or plasma by nucleic acid amplification methods is important for confirming the diagnosis of HCV, distinguishing active from resolved infection, assessing the virologic response to therapy, and screening the blood supply. These tests are incorporated into diagnostic algorithms for HCV proposed by the National Institutes of Health,36 the CDC,37 European Association of the Study of the Liver,38 and National Academy of Clinical Biochemistry.39

The detection of HCV RNA in the plasma or serum is the earliest marker of infection, appearing 1 to 2 weeks after infection and weeks before the appearance of alterations in liver enzyme levels or anti-HCV antibodies. Approximately 80% of individuals infected with HCV will be chronically infected with the virus. In antibody-positive individuals, HCV RNA tests can distinguish active from resolved infections. In patients with a high pretest probability of infection, a positive serological screening test is usually "confirmed" with a qualitative test for HCV RNA rather than the supplemental recombinant immunoblot assay (RIBA). This strategy is cost-effective and more informative than using the RIBA to confirm positive antibody screening tests in a diagnostic setting. It is important to remember that the RIBA confirms the presence of antibody, whereas an HCV RNA test detects the presence of the virus.

The CDC recently developed guidelines for HCV antibody testing in which the signal to cutoff (s/co) ratio of the serological screening test can be used effectively to determine which screening test results need supplemental testing for confirmation.40 Screening enzyme-linked immunoassay results with s/co ratios >3.8 predict a RIBA-positive result >95% of the time in all populations tested. Consequently, supplemental testing is required only for those screening tests with low s/co ratios. Either a RIBA

or an HCV RNA test can be used for confirmation, but all negative HCV RNA tests should be followed by a RIBA since a single negative RNA test does not rule out infection. Confirmation of an HCV antibody screening test is important to ensure that further clinical evaluation is limited to those patients who are truly antibody positive and to reduce psychological stress on patients who test falsely positive for antibody.

HCV RNA testing also is helpful for the diagnosis of infection in infants born to HCV-infected mothers, due to persistence of maternal antibody, and in immuno-compromised or debilitated patients who may have blunted serological responses. A qualitative HCV RNA test also should be used for patients suspected of having an acute infection and in patients with hepatitis of no identifiable cause.

Qualitative HCV RNA tests are the most reliable means of identifying patients with active HCV infection. A negative HCV RNA test in a serologically positive individual may indicate that the infection has resolved or that the viremia is intermittent. Up to 15% of chronically infected individuals may have intermittent viremia and, as a result, a single negative HCV RNA determination may not be sufficient to exclude active infection when the index of clinical suspicion is high.41 In these individuals a second specimen should be collected and tested.

IFN-based regimens, either IFN in combination with ribavirin or IFN monotherapy for those patients who cannot tolerate ribavirin, are the standard treatment for patients with chronic HCV infection. Because these regimens produce sustained virological responses in only about half of the patients treated, are of long duration (24 to 48 weeks), and are associated with significant adverse events, early identification of patients who are likely or unlikely to respond to treatment is desirable.

Early clearance of HCV RNA as assessed with a sensitive assay or a rapid decline in HCV RNA levels during the early treatment period is predictive of sustained virological response among patients receiving either standard or pegylated IFN alone, and pegylated IFN plus ribavirin.42,43 Patients who have detectable or less than a 2 log10 decrease in viral RNA levels after 12 weeks of treatment have a minimal chance of achieving a sustained virological response (negative predictive value of 98%).

In contrast, early viral kinetics are less predictive of sustained virological response among patients treated with standard IFN and ribavirin because late responses are seen in some patients. Thus, discontinuing therapy in patients based on a positive HCV RNA test early in treatment would deny the treatment to a substantial number of patients who would achieve a sustained response. The virological response to combination therapy with IFN and ribavirin should be assessed after 24 weeks of therapy with a sensitive (<50IU/ml) qualitative assay for HCV RNA.44 Individuals with a positive qualitative RNA test after week 24 of therapy are considered treatment failure and therapy is discontinued. Therapy also can be safely stopped at 24 weeks in patients infected with HCV genotypes 2 and 3 who have a negative HCV RNA test,because there is no benefit to longer therapeutic regimens. However, an additional 24 weeks of therapy is recommended for patients infected with other genotypes, even with a negative qualitative HCV RNA test. In addition, a qualitative HCV RNA test should be performed 24 weeks after completion of treatment in all patients to establish if a sustained virological response was achieved regardless of the treatment regimen.

The use of anti-HCV antibody tests to screen the blood supply has dramatically reduced the risk of transfusion-associated HCV infection in developed countries. The risk in the United States from blood that is negative for anti-HCV antibodies is less than 1 in 103,000 transfused units.45 To drive the risk of infection even lower, blood donor pools are currently tested for the presence of HCV RNA.46 The serologic screening tests for HCV have a 70-day window period of seronegativity, and antigen detection tests are not yet available for blood bank screening. It is estimated that HCV RNA testing will reduce the detection window by 25 days and reduce the number of infectious units from 116 to 32 per year.47

An enzyme immunoassay (EIA) for the detection and quantitation of total HCV core antigen in serum has recently been developed and is available as a research-use-only test (Ortho Trak C, Ortho Clinical Diagnostics, Raritan, NJ). This test significantly shortens the serologi-cally silent window period using seroconversion panels, and its performance correlates closely with RNA detection tests in blood donors.48,49 However, the analytical sensitivity is less than most RNA tests, at approximately 20,000 IU/ml. The analytical sensitivity of the core antigen test is too high to be used in the monitoring of late events during and after treatment.50 Antigen detection may represent a cost-effective alternative to HCV RNA testing to distinguish active from resolved infections in resource-poor settings.

HCV viral load testing is useful in pretreatment evaluations of patients being considered for therapy, since a viral load of less than 2 x 106 copies/ml (800,000IU/ml) is one of several predictors of achieving a sustained virological response.51,52 Other factors associated with achieving a sustained response to therapy include the absence of cirrhosis, age less than 40 years, female gender, white race, and viral genotype other than 1. Viral load testing also can be used in an early assessment of viral kinetics in patients treated with IFN alone or with pegylated IFN plus ribavirin.42,43 Patients who fail to achieve less than a 2 logi0 decline in viral load with treatment have little chance of achieving a sustained virological response to these therapies.

HCV viral load does not predict disease progression and is not associated with severity of liver disease.53 This is in sharp contrast to HIV-1,in which the viral load is the principal factor determining the rate of disease progression. Monitoring HCV viral load in untreated patients is not warranted and should be discouraged.

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