EVALUATION OF BRANCHED DNA SIGNAL AMPLIFICATION FOR THE DETECTION OF HEPATITIS-C VIRUS-RNA

There is an increasing need for a practical assay to measure HCV RNA t o assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response, This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). T he bDNA assay uses a microtitre well format and a series of capture, t arget and amplification probes that bind RNA to the well and then succ essively bind oligonucleotides to the RNA and branched DNA molecules t o the oligonucleotides, Enzyme-labelled probes are bound to the arms o f the bDNA and light output from a chemiluminescent substrate is direc tly proportional to the amount of starting HCV RNA. Appropriate standa rds provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, c oded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all si x infectious sera, Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commerc ial seroconversion panels, In acute cases, 10(7)-10(8) molecular equiv alents per mi (eq per mi) of HCV RNA were detected prior to peak alani ne aminotransferase (ALT) activity and then rapidly declined to non-de tectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chroni c course was characterized by diminished, fluctuating and sometimes no n-detectable levels of HCV RNA, In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when a ssayed by PCR, In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship b etween viral replication and subsequent hepatocellular injury, In test ing 50 blood donors whose anti-HCV reactivity was confirmed by a recom binant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81 %), while PCR was positive in 45 (90%); the overall concordance betwee n bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive dono r samples was 96%. Lastly, bDNA showed the loss of HCV RNA in six out of six evaluable patients who had complete biochemical responses to in terferon; five out of six non-responders also showed appreciable decli nes in HCV RNA level, but in only two did HCV RNA drop below the detec tion limit; these two cases remained PCR positive, Seventeen placebo-t reated patients did not lose HCV RNA by either bDNA or PCR, Hence the bDNA assay is a practical means to measure HCV RNA in a variety of cli nical settings, Although it is not as sensitive as PCR, it has greater specificity, is directly quantitative, and can be used in any routine laboratory that can perform microwell EIAs. This simplified quantitat ion may be of particular benefit in evaluating the probability of HCV transmission and the response to anti-viral therapy.