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-ch ain-reaction(PCR)-based semi-quantitative detection of HDV RNA. The se miquantification method used was based on the appearance of a positive amplification signal as a function of the number of PCR cycles. By am plifying 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 m onths. All samples were amplified in parallel for 20 and 30 PCR cycles . Analysis of HDV cDNA after ethidium bromide/agarose gel electrophore sis and after molecular hybridization (100 times more sensitive than g el 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 t reated 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 HD V RNA decrease or disappearance was evidenced in the serum at the 6th month; however, viral replication recurred at a higher level at the 12 th month. Finally; the HDV genome was no longer detected during therap y in the final 4 patients. These results were compared to clinical and biological data. The level of alanine aminotransferase (ALAT) paralle led HDV RNA gradation in 15 samples from 5 patients. However, in 2 pat ients, 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 chr onic 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 o f only two PCR experiments and quickly provides information on the lev el of HDV viraemia.