SPECIFIC, SENSITIVE, AND RAPID ASSAY FOR HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1 POL MUTATIONS ASSOCIATED WITH RESISTANCE TO ZIDOVUDINE AND DIDANOSINE

The effectiveness of antiretroviral therapy may be limited by the deve lopment of human immunodeficiency virus type 1 (HIV-1) resistance. Mon itoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status, Our obj ective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which i s simple to perform, In our assay the DNA of HIV-1 pol was amplified b y PCR using two sets of nested oligonucleotide primers, Mutations of r everse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically, The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing, To evaluate the limits o f the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR, The assay was sensitive and specific for a ll specimens except when mutations occurred within 2 bases on either s ide of the ligation site, In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations CRT aa 74, 41, 70, an d 215) associated with HIV-1 resistance to ddI and ZDV.