POLYMERASE-CHAIN-REACTION-BASED SEMI-QUANTIFICATION OF HEPATITIS-D VIREMIA IN PATIENTS TREATED WITH HIGH-DOSES OF ALPHA-2B INTERFERON

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 semiquantification 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. Finally; the HDV genome was no longer detected during therapy in the final 4 patients. These results were compared to clinical and biological data. The level of alanine aminotransferase (ALAT) paralleled HDV RNA gradation in 15 samples from 5 patients. However, in 2 patients, HDV-RNA was negative at the 6th month, before the normalization of ALAT. In the serum of another patient, HDV RNA was still detected when the ALAT level was normal. This study shows the usefulness of HDV PCR in appreciating the potential benefits of alpha 2b-IFN during chronic hepatitis D infection. However, long-term improvement was present in only one patient in whom a negative HDV PCR result was associated with loss of HBs-Ag. This simple approach uses complementary results of only two PCR experiments and quickly provides information on the level of HDV viraemia.