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M94A3317.TXT
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1994-10-25
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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).