Document 3317
 DOCN  M94A3317
 TI    The microtiter point mutation assay to detect drug resistance in
       HIV-seropositive patients treated with AZT.
 DT    9412
 AU    van der Feltz M; de Haas C; de Graaf L; Borleffs JC; Visser MR; Verhoef
       J; University Hospital, Dept. of Internal Medicine, Immunology and; Inf.
       Dis., Utrecht, The Netherlands.
 SO    Int Conf AIDS. 1994 Aug 7-12;10(1):103 (abstract no. PA0032). Unique
       Identifier : AIDSLINE ICA10/94369258
 AB    BACKGROUND: After prolonged therapy of HIV-seropositive patients with
       zidovudine (AZT), drug resistant virus strains emerge. Five point
       mutations of the viral reverse transcriptase are responsible for various
       degrees of drug resistance. A rapid, simple method to detect the
       proportions of wild type and mutant sequences in a patient can be of
       significant value to evaluate the benefit of long term treatment. It can
       also be of value in clinical trials with new antiretroviral drugs to
       screen for rapidly appearing point mutations causing resistance.
       METHODS: The microtiter point mutation assay according to Kaye et al.
       (J. of Med. Vir. 1992) was used. A double stranded biotinylated PCR
       product is captured on a streptavidin coated microtiter well and
       denatured to a single strand. A probe with its 3' end one base upstream
       of the point mutation under analysis (X) is annealed and a single
       labelled dNTP (Y) is used to extend the probe if Y is complementary to
       X. The extended probe is denatured away from the target and added to a
       scintillation cocktail for counting. Wild type and mutant strain
       percentages can be calculated. Fourteen pairs of patients' samples were
       tested in the assay: one sample before the use of AZT, the other one
       after approximately one year's use of AZT. Samples of a chronically
       infected cell line containing a wild type sequence (pre A012) and one
       containing the five mutations (post A012) were used as controls. All
       assays were performed in duplicate. RESULTS: At codons 70 and 215, a
       shift from predominantly wild type strains (less than 15% mutated
       sequences) to a mixture of wild type and mutant strains (30-100% mutated
       sequences) appeared in the majority of the patients' samples after one
       year's treatment with AZT. At codon 67 and 219, the wild type strain
       persisted after therapy in all but 2 patients. At codons 41, the
       proportion of mutant strains increased after treatment. Our results are
       in agreement with the literature in which codons 70 and 215 have been
       described to mutate rapidly after commencement of therapy. CONCLUSION:
       The microtiter point mutation assay is a rapid and simple method to
       screen for point mutations causing drug resistance and to quantitate the
       proportion of mutated viral sequences.
 DE    Drug Resistance, Microbial/GENETICS  *Genetic Techniques  Human
       HIV/*DRUG EFFECTS/ENZYMOLOGY/*GENETICS  HIV Seropositivity/*DRUG
       THERAPY/*MICROBIOLOGY  *Point Mutation  Polymerase Chain Reaction
       Reverse Transcriptase/GENETICS  Time Factors  Zidovudine/*THERAPEUTIC
       USE  MEETING ABSTRACT

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