Although these newer tests for anti-HCV have improved diagnostic accuracy by increasing sensitivity and narrowing the window period between infection and viral detection, they are still not absolutely dependable for identifying hepatitis C. Moreover, although a positive test result generally indicates active infection, it does not distinguish between current and previous disease and therefore is of no benefit in assessing recovery after antiviral treatment. Identification of the hepatitis C virus itself would clearly be more useful. Much progress has been made with procedures that detect viral nucleic acids by the use of molecular or signal amplification technologies, and the presence of HCV RNA can now be determined at levels as low as only a few molecules. In addition, viral concentrations can be measured in clinical specimens with both technologies, albeit with differing degrees of sensitivity and practicality. This not only aids diagnosis but also allows the use of accrued information to predict outcome of disease as well as the prospect of response to antiviral treatment.

The most widely used viral nucleic acid assays are RT-PCR (reverse transcription of the viral RNA followed by amplification of complementary DNA by the polymerase chain reaction [PCR]) [8] and the bDNA assay (capture of the viral RNA by virus-specific probes followed by hybridization to branched-DNA molecules, which are in turn detected by an enzyme-chemiluminescent substrate system) [9]. The former assay can be done in its original, simpler version, in which it provides qualitative information on the presence of HCV, or it can be made quantitative by the use of modified versions such as quantitative competitive PCR [10]. This involves the addition of known amounts of similar but different RNA molecules to the reaction tubes. The extraneous RNA is transcribed and amplified simultaneously with the viral nucleic acid, serving as both an internal control and a standard for quantitation. The current problems with quantitative PCR are its high cost and laborious nature and the lack of standardization. Nevertheless, when the assay is done under optimal conditions, extremely important information is gleaned.

In this issue, the pivotal role of HCV RNA identification as a diagnostic and prognostic tool is addressed by Gretch and colleagues [11]. They examine quantitative PCR and the bDNA assay and assess the utility of the tests among four different groups of patients, including prospective blood donors, patients who are attending clinics and have varying degrees of liver disease, patients receiving renal dialysis, and HCV-infected liver transplant recipients. On the basis of the data from this meticulously conducted study, the authors conclude that the two tests are well correlated in all groups of patients, that quantitative PCR has a broader linearity range than the bDNA assay, and that the sensitivity of quantitative PCR is approximately four orders of magnitude greater than that of the bDNA assay. Gretch and colleagues point out that during interferon- treatment, HCV levels may decrease to those that are not detected by the bDNA method but that can be detected by RT-PCR; they conclude that 12% to 15% of nonimmunosuppressed persons with hepatitis C viremia are not identified by the bDNA assay. This finding indicates that despite apparent treatment-induced virus loss, HCV RNA may still persist and be associated with biochemical relapse. Thus, nonreactivity by the bDNA assay cannot be used as a criterion for virus clearance. The authors also note the absence of linearity in the bDNA assay when the viral load is high; the significance of this observation is not yet clear.

Other methods used to identify the virus, such as cell culture systems or electron microscopy, have been unsuccessful. Thus, the practicing clinician can currently use the following diagnostic tools: 1) measurement of ALT activity, which merely defines existing liver disease and correlates poorly with HCV-related disease activity, particularly in the instance of chronic hepatitis C; 2) liver biopsy, an invasive procedure that is subject to sampling errors and morphologic variability; and 3) a series of serologic assays that include ELISA, RIBA, RT-PCR, bDNA, and quantitative PCR. The latter assay, although highly sensitive, is too complex and labor-intensive for most routine hospital laboratories. Given the remaining diverse and confusing array of potential diagnostic measures, how should the practicing clinician evaluate the patient with suspected HCV infection? Should the choice of test be tailored to the clinical circumstances?

Serologic assays now seem to serve two roles in evaluating persons suspected of having HCV infection: diagnosis of the disease and provision of data helpful in predicting treatment response or even treatment consideration. We present the following thoughts on these two roles. First, measurement of ALT activity remains important and, indeed, essential in evaluation. However, it appears less useful for HCV infection than for other forms of viral hepatitis because of the inconsistent correlation between biochemical and histologic indices of severity in both acute and chronic hepatitis C [12]. Significant histologic abnormalities may be found even among HCV-infected persons with normal serum enzyme levels [13].

Second, despite its shortcomings, liver biopsy remains important in evaluating persons with chronic but not acute hepatitis C. Not only are biopsy specimens useful baseline measures for judging disease severity and progression, but they also provide useful prognostic information on natural history and the potential for treatment response. In patients with hepatitis C, the presence of cirrhosis diminishes the likelihood for success [14].

Third, we believe that the choice of serologic assay should depend on the clinical circumstance. For suspected acute hepatitis C, only RT-PCR has diagnostic relevance. Early diagnosis may assume greater meaning if increased consideration is given to early antiviral treatment in the hope of arresting progression from acute to chronic hepatitis C. In the absence of available PCR technology, anti-HCV can be sought in convalescent serum samples. For chronic hepatitis C, the initial approach is to seek anti-HCV using the ELISA assay. Thereafter, evaluation can vary with clinical presentation.

Among patients with typical hepatitis symptoms or sustained elevated serum enzyme levels, ELISA reactivity almost always indicates true HCV infection, particularly if the ratio between the sample value and the cutoff value exceeds 2.5 (manufacturer's positive cutoff value, more than 1.0) (personal observation). If treatment is now contemplated, a baseline bDNA assay can be useful because some evidence suggests that the lower the HCV RNA concentration, the higher the response; in one study, the best response was seen in persons with HCV RNA levels less than 4 mEq/mL [15]. If ALT values do not return to normal within 1 to 3 months of starting treatment, the likelihood of later success is low and treatment may be discontinued. If the values do return to normal, the bDNA assay can be repeated; if it is still reactive, the likelihood of a sustained response after treatment completion is low. An increase in treatment dose might therefore be considered. If the bDNA assay is nonreactive, loss of viremia cannot be assumed until confirmed by RT-PCR, as pointed out by Gretch and colleagues. If RT-PCR is reactive, a sustained response is unlikely, and a dose increase can now be considered. If RT-PCR is nonreactive, standard treatment will probably be successful. Success is defined as persisting absence of HCV RNA for at least 6 months after therapy is discontinued.

Persons with persistently normal ALT values and ELISA reactivity, particularly if they have no symptoms or have an associated hypergammaglobulinemia, require supplementary RIBA testing. A positive result indicates true reactivity; a negative result, false-positivity. An indeterminate result requires confirmation by PCR. Few investigators currently treat anti-HCV-reactive persons who have normal enzyme levels. If these patients are treated, however, RT-PCR becomes the only way to define success or failure.

The rapid technologic advances now occurring are bringing us to the brink of a gold standard panel of tests for hepatitis C. The greatest problems currently associated with virologic assessment are poor standardization, the need for dedicated work space, and the absolute necessity of strict adherence to procedures that avoid contamination. Also important, as pointed out by Gretch and colleagues, is that careful work and proper analysis of acquired data must continue so that the best possible interpretation is applied to the results. Indeed, the rapidity of progress in this area suggests that recommendations made today may well become outdated as new data become available.