SEMIQUANTITATIVE ASSESSMENT OF PRECORE STOP-CODON MUTANT AND WILDTYPEHEPATITIS-B VIRUS DURING THE COURSE OF CHRONIC HEPATITIS-B USING A NEW PCR-BASED ASSAY

In most patients with chronic hepatitis B positive for antibodies (ant i-HBe) to HBe antigen (HBeAg), a pre-core mutant hepatitis B virus (HB V) with a point-mutation at nt. 1896 can be isolated. Clinical signifi cance of the mutant virus in chronic hepatitis B is not proven yet, an d screening of large numbers of sera during different clinical courses of numerous patients is necessary. We therefore aimed to develop a fa st and reliable assay, that allows to discriminate wildtype from nt. 1 896 G-->A mutant HBV and to determine the ratio of mutant and wildtype HBV in patients' sera. A mutation specific polymerase chain reaction (ms PCR) with new primers served to distinguish nt. 1896 G-->A mutant from wildtype HBV. This msPCR proved to be more sensitive and specific than similar assays described previously. When compared to a dilution series of a cloned HBV-DNA standard, the amount of wildtype and nt. 1 896 G-->A mutant HBV could be determined semiquantitatively. 10(2) to 10(7) copies of each HBV-DNA (equivalent to 10(5) to 10(10) copies of HBV-DNA/ml patients' serum) could be amplified with steadily increasin g signals. MsPCR proved to be specific as 10(7) copies did not give an amplification signal if they did not match the respective primer pair used. In a mixed population of mutant and wildtype virus, msPCR allow s to detect even a low amount of the minor HBV strain (0.1-0.01%, of t he viral population) and to determine the ratio of wildtype and mutant HBV. MsPCR is as fast and convenient to perform as an unmodified PCR. It requires careful performance to avoid contamination but no specifi c equipment. Clinical usefulness of msPCR was demonstrated when the ra tio of wildtype to mutant HBV was determined in 86 sera collected duri ng 3 to 7.5 years follow up of 9 patients suffering from anti-HBe posi tive chronic hepatitis B. We conclude that this assay conveniently all ows to study patients with chronic hepatitis B in order to detect and follow-up the emergence of pre-core stop-codon mutant HBV in correlati on to the clinical course.