Polymerase-chain-reaction-based semi-quantification of hepatitis D viraemia in patients treated with high doses of...

To study the antiviral efficacy of high doses of alpha 2b interferon (alpha 2b-IFN) for chronic hepatitis D treatment, we used polymerase-chain-reaction(PCR)-based semi-quantitative detection of HDV RNA. The semi-quantification method used was based on the appearance of a positive amplification signal as a function of the number of PCR cycles. By amplifying dilutions (10(-1)-10(-8)) of an HDV-positive woodchuck liver RNA, we confirmed that exponential amplification efficacy occurred at between 20 and 30 cycles. Positive signals were observed from dilution 10(-2) (gel electrophoresis after 20 cycles of PCR) to dilution 10(-7) (hybridization after 30 cycles of PCR). To characterize the HDV RNA level in sera of 8 patients treated with alpha 2b-IFN (10 MU/3 times a week) for 1 year, we extracted RNA from serum samples taken every 6 months. All samples were amplified in parallel for 20 and 30 PCR cycles. Analysis of HDV cDNA after ethidium bromide/agarose gel electrophoresis and after molecular hybridization (100 times more sensitive than gel analysis), enabled us to grade the signals observed from negative to positive as 1+, 2+, 3+ and 4+, with all results being positive. Three types of evolution of HDV viraemia were evidenced among the 8 treated patients. HDV replication continued to occur at a high level at the 6th and 12th month in 2 patient sera. For 2 other patients, an HDV RNA decrease or disappearance was evidenced in the serum at the 6th month; however, viral replication recurred at a higher level at the 12th month.(ABSTRACT TRUNCATED AT 250 WORDS)