Comparison of a point mutation assay with a line probe assay for the detection of the major mutations in the HIV-1 reverse transcriptase gene associated with reduced susceptibility to nucleoside analogues

This study compares the performance of a line probe assay (LiPA) for the de tection of the major mutations associated with reduced sensitivity to nucle oside analogues with a well characterised point mutation assay (PMA). Plasm a samples obtained from patients in a trial of four reverse transcriptase i nhibitors (MRC Quattro Trial) were tested by both LiPA and PMA at baseline, 32nd and 64th weeks for the presence of drug resistance associated mutatio ns in the reverse transcriptase (RT) gene. HIV-1 RNA was extracted from pla sma by the Boom method and amplified by RT-PCR prior to being tested by LiP A or PMA. Assay discrepancies were further investigated by sequencing of th e RT gene. Of 275 samples available from 98 trial subjects, 246 samples wer e successfully amplified by PCR and analysed by LiPA and PMA for six mutati ons. Of the 1476 individual codons analysed, LiPA successfully assayed 1444 (97.8%) and PMA gave a result with 1418 (96.1%). LiPA failed to give a res ult for 32 codons from 22 samples and PMA failed with 58 codons from 38 sam ples. Gross differences between the two assays, in which one scored a codon as wild-type only and the other as mutant only or vice versa, occurred at 28 codons analysed (1.9%) representing 26 samples from 20 subjects. Sequenc ing of 22 of the 26 samples confirmed the LiPA result in nine cases, the PM A result in 11 and detected a novel variant at codon 215 in four cases. The PMA and LiPA approach to the detection of the major mutations that are gen otypically associated with reduced sensitivity to nucleoside analogues can correctly detect mutations in 97% of the cases. (C) 2000 Elsevier Science B .V. All rights reserved.