Five unique human hepatitis viruses have been identified (1-5). The hepatitis A virus and hepatitis E virus are enterically transmitted RNA viruses that do not cause chronic liver disease. In contrast, the hepatitis B virus, hepatitis C virus and hepatitis D virus (HBV, HCV and HDV, respectively) are parenterally transmitted and cause chronic infection. They are dangerous contaminants of the blood supply. Recently tests have become readily available for testing for HBV in blood, allowing for the screening for this pathogen and the elimination of infected samples from the blood supply (6).
Concomitant with the availability of the HBV test came an increase in the proportion of cases of post-transfusion hepatitis due to non-A, non-B (NANB) agents. Until recently, there was no test available for the detection of the NANB agents. The principal NANB agent, HCV, was recently identified by molecular cloning of segments of the HCV genome (3). HCV is an RNA virus related to human flaviviruses and animal pestiviruses (7,8).
Prospective studies of selected counties in the United States by the Centers for Disease Control (CDC) indicate that approximately 170,000 new cases of NANB/HCV infection occur yearly (9). At least 50% of these infections appear to progress to chronic liver disease. Severe sequelae include the development of decompensated cirrhosis necessitating liver transplantation, and development of hepatocellular carcinoma (10,11).
The positive-stranded RNA genome of the HCV contains approximately 10,000 nucleotides. The HCV genome acts as a long open reading frame (ORF) capable of encoding a 3,010 amino acid polyprotein precursor from which individual viral proteins, both structural and nonstructural, are produced (7,12-14). There are at least 324 nucleotides at the 5'-end of the ORF which have not yet been shown to encode for protein. Thus, this sequence is referred to as the 5'-non-coding region (7,12-17). Several research groups have reported the nucleotide sequence of either the whole HCV genome or specific subgenomic regions (7,12-22). Comparison of these sequences demonstrates variations in the structural and nonstructural regions (ranging from 9-26%) among different HCV strains. In contrast, the sequences of the 5'-non-coding region appear to have a homology of approximately 99% among different strains (16,17). The 5'-non-coding region also has substantial homology (45-49%) with the equivalent region of animal pestiviruses (7).
Two major techniques are currently used to detect HCV infection. The first technique detects antibody produced in response to HCV infection (anti-HCV) (23-28). Since multiple weeks are required for infected patients to develop detectable IgG antibody against HCV antigens, this test is useless in the detection of acute HCV infection. Moreover, studies indicate that antibody testing is associated with both false positive and false negative results (29).
These shortcomings in the original assays have spurred development of newer supplemental antibody tests for the diagnosis of HCV infection (30). Preliminary results with supplemental assays indicate a decrease in the frequency of false-positive and negative results. However, false-positive and -negative results still occur and supplemental tests remain unsuitable for detection of acute infection (31).
The second technique, detection of HCV RNA by an RNA polymerase chain reaction (PCR), has been limited to research use. The HCV PCR evaluates infection by detecting HCV RNA in blood or tissue extracts through reverse transcription and cDNA amplification (7,32-41). HCV PCR represents a sensitive, direct technique but requires meticulous care (7) to prevent false positive and negative results. The HCV PCR technique, in contrast to antibody tests, can detect circulating HCV RNA during acute infection.
The original HCV PCR tests used primers specific for sequences in the non-structural region of the HCV genome (32-36). Subsequently, HCV PCR has been performed using several primers for the 5'-non-coding region in the genome (37,39). In our laboratory we have established HCV PCR for both the nonstructural and 5'-non- coding regions. Our comparative results indicate that the HCV PCR from the 5'-non-coding region is more sensitive in detecting HCV infection (41).
Despite the success of HCV PCR, the technique has many inherent limitations. First, it is time consuming, expensive and dependent upon meticulous technique. The exquisite sensitivity of PCR makes false positive results due to contamination with exogenous HCV RNA a constant concern (42). Moreover, the variation in both the reverse transcription of HCV RNA to CDNA and the amplification of cDNA make the HCV PCR difficult to quantitate (38,40,42). Recent attempts to overcome these obstacles have resulted in, at best, semi-quantitative assays (38). More importantly, in our experience the efficiency of HCV PCR depends in large part on the specific primers employed. Not only have standards for primers not been developed, but polymerases employed in PCR have different efficiencies. Thus, it will likely be difficult to compare PCR results among different laboratories.
To overcome the limitations of current antibody and HCV PCR techniques for detection of HCV infection, it is desirable to develop a test which is highly sensitive, specific, affordable and applicable to the testing of large populations of patients or blood donors. The optimal test would be capable of detecting both acute and chronic infection. Moreover, it would be quantitative to provide information regarding both natural history and the efficacy of current or future antiviral therapies. It would be capable of uniform results. These prerequisites can be fulfilled by a technique to directly detect HCV RNA.