Polymerase-chain-reaction-based semi-quantification of hepatitis D viraemia 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 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)
Dény
P
P
Laboratoire de Bactériologie-Virologie, CHU Avicenne, UFR de Bobigny, Université Paris XIII, France.
Fattovich
G
G
Le Gal
F
F
Giustina
G
G
Lecot
C
C
Morsica
G
G
Poinsot
H
H
Alberti
A
A
Bréchot
C
C
D000328:Adult; D001483:Base Sequence; D005260:Female; D003699:Hepatitis D / Q000150:complications / Q000188:drug therapy*; D003698:Hepatitis Delta Virus / Q000187:drug effects; D006521:Hepatitis, Chronic / Q000188:drug therapy; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000627:therapeutic use*; D008103:Liver Cirrhosis / Q000150:complications; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D011994:Recombinant Proteins; D014766:Viremia / Q000188:drug therapy*; D014779:Virus Replication / Q000187:drug effects
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins