Document 1095
 DOCN  M94A1095
 TI    Limiting dilution PCR to detect changes in the RT of HIV-1 from patients
       on long term AZT treatment.
 DT    9412
 AU    Stein CA; Levantis P; Goh B; Hillman R; Oxford JS; London Hospital
       Medical College, UK.
 SO    Int Conf AIDS. 1994 Aug 7-12;10(2):21 (abstract no. 378A). Unique
       Identifier : AIDSLINE ICA10/94371480
 AB    OBJECTIVES: To investigate any amino acid (aa) changes in the Reverse
       Transcriptase (RT) enzyme of HIV-1 in sequential samples (up to
       twelve/subject) from patients infected with HIV-1 and treated with AZT
       or ddI. To analyse the distribution of proviral DNA sequences, utilising
       a end point dilution polymerase chain reaction (PCR) method to detect
       single RTs with multiple samples at every time point. To relate changes
       in the aa sequence to the clinical status of a patient and to a
       crystallographic model of RT. MATERIALS AND METHODS: We investigated the
       clinical and molecular history from five HIV+ patients since the onset
       of AZT treatment. Serial samples, taken every 3 months from treatment
       time 0 up to > 2.5 years, were analysed. 10 ml of heparanised blood were
       taken and peripheral blood mononuclear cells (PBMC) separated by density
       centrifugation. DNA was phenol-chloroform extracted from the PBMC and
       amplified by a highly sensitive, high yield limiting dilution double
       PCR. The products, representing a singular HIV-1 RT molecule were then
       analysed by direct sequencing. RESULTS AND CONCLUSIONS: We observed the
       development of a plurality of mutations in a broad panel of different
       HIV-1 RT molecules in each patient over time and at each time point.
       Each patient carried a variety of different HIV-1 RTs in their genome at
       any time. The amino acid changes concentrated in two small domains on
       the RT, between aa position 63-90 and between position 180-220. It was
       of particular interest that AZT sensitive sequences were still detected
       after more than fifteen months of treatment. In addition to the most
       common mutations, we have observed a number of other changes that could
       be essential for the development of drug resistance. The aa changes have
       been fitted to the crystallographic RT model of the group of Arnold et
       al.
 DE    Didanosine/PHARMACOLOGY/*THERAPEUTIC USE  Drug Resistance,
       Microbial/GENETICS  Human  HIV-1/DRUG EFFECTS/ENZYMOLOGY/*GENETICS
       Mutation  Polymerase Chain Reaction/*METHODS  Reverse
       Transcriptase/*GENETICS  Zidovudine/PHARMACOLOGY/*THERAPEUTIC USE
       MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).