Detection of Mutations Associated with Zidovudine Resistance in Human Immunodeficiency Virus by Use of the Polymerase Chain Reaction

Abstract

A sensitive and specific polymerase chain reaction (PCR)-based assay was developed for four mutations in the reverse transcriptase gene of human immunodeficiency virus type 1 that have been associated with zidovudine resistance. These mutations were correlated in 366 specimens with zidovudine chemotherapy and resistance. Mutations at these four codons were detected only after zidovudine therapy. The usual sequence of appearance of mutations was codons 215, 70, 67, and 219, although individual variations occurred. The degree of resistance was proportional to the number of mutations present, although variable susceptibilities with identical patterns of mutations suggested the likelihood that additional mutations contribute to resistance. The existence of both phenotypic and genotypic mixtures was documented as was the occasional selection of subpopulations with passage of virus in vitro. The many complexities of zidovudine resistance render the assay of limited use for application to individual patients; however, it could prove useful for correlating disease or therapy with the emergence of resistance.

Effects of discontinuation of zidovudine treatment on zidovudine sensitivity of human immunodeficiency virus type 1 isolates

“…For some patients, however, no change at codon 41 is found. A similar pattern was observed during treatment of a group including both symptomatic and asymptomatic individuals (13). In vitro serial passage of the HIV laboratory strain HXB-2 in the presence of gradually increasing concentrations of zidovudine gives a similar order of appearance of mutations (9).…”

Genotypic Testing for Human Immunodeficiency Virus Type 1 Drug Resistance

“…The NEMs are selected primarily in patients treated with zidovudine or stavudine alone or in combination with other NRTIs (27,49,169,203,232,266,285,291,305,311,335,353 (261). There are few data on the development of NEMs in patients receiving zalcitibine or tenofovir without other NRTIs.…”

Effects of Specific Zidovudine Resistance Mutations and Substrate Structure on Nucleotide-Dependent Primer Unblocking by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

“…Mutations at codons 41, 67, 70, 210, 215, and 219 in the HIV-1 RT gene result in resistance of HIV-1 to AZT in cell culture assays (29). Substitutions of phenylalanine and tyrosine for threonine at position 215 (T215F and T215Y) are the predominant mutations observed in vivo and are considered the most important for the resistance phenotype (16,17,25).The inhibitory effect of incorporating a chain-terminating nucleotide analogue can be partially relieved by a reaction catalyzed by RT in which the terminating nucleotide is removed from the 3Ј end of a DNA chain by transfer to a nucleotide di-or triphosphate, producing an unblocked DNA chain and dinucleoside polyphosphate with the chain terminator linked to the nucleotide acceptor through a tri-or tetraphosphate chain (19,21). HIV-1 RT can also transfer the chain-terminating residue to pyrophosphate (PP i ), regenerating the triphosphate form of the chain terminator (1, 5, 10, 23).…”

“…Mutations at codons 41, 67, 70, 210, 215, and 219 in the HIV-1 RT gene result in resistance of HIV-1 to AZT in cell culture assays (29). Substitutions of phenylalanine and tyrosine for threonine at position 215 (T215F and T215Y) are the predominant mutations observed in vivo and are considered the most important for the resistance phenotype (16,17,25).…”