Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse 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 of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV.

[1]  E. Walter,et al.  HIV-1 sensitivity to zidovudine and clinical outcome in children , 1992, The Lancet.

[2]  H. Gendelman,et al.  Biological and biochemical characterization of a cloned Leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus , 1986, The Journal of experimental medicine.

[3]  G L Drusano,et al.  A controlled trial of early versus late treatment with zidovudine in symptomatic human immunodeficiency virus infection. Results of the Veterans Affairs Cooperative Study. , 1992, The New England journal of medicine.

[4]  S. Spector,et al.  Development and significance of zidovudine resistance in children infected with human immunodeficiency virus. , 1993, The Journal of pediatrics.

[5]  S D Kemp,et al.  Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). , 1989, Science.

[6]  P. Andrews,et al.  A universal nucleoside for use at ambiguous sites in DNA primers , 1994, Nature.

[7]  P. Nara,et al.  Quantitative infectivity assay for HIV-1 and -2 , 1988, Nature.

[8]  B. Larder,et al.  Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. C. J. D. D. R. C. R. Wilbanks.  Zidovudine for the prevention of HIV transmission from mother to infant. , 1994, MMWR. Morbidity and mortality weekly report.

[10]  Ann Marie Swart,et al.  Concorde: MRC/ANRS randomised double-blind controlled trial of immediate and deferred zidovudine in symptom-free HIV infection , 1994, The Lancet.

[11]  D. Richman,et al.  Characterization of genetic variation and 3'-azido-3'-deoxythymidine- resistance mutations of human immunodeficiency virus by the RNase A mismatch cleavage method. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Loveday,et al.  A microtitre format point mutation assay: Application to the detection of drug resistance in human immunodeficiency virus type‐1 infected patients treated with zidovudine , 1992, Journal of medical virology.

[13]  M A Fischl,et al.  The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind, placebo-controlled trial. , 1987, The New England journal of medicine.

[14]  D. Katzenstein,et al.  Zidovudine susceptibility testing of human immunodeficiency virus type 1 (HIV) clinical isolates. , 1993, Journal of virological methods.

[15]  S D Kemp,et al.  Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes. , 1991, AIDS.

[16]  M. Gonda,et al.  Simple, rapid, quantitative, syncytium-forming microassay for the detection of human immunodeficiency virus neutralizing antibody. , 1987, AIDS research and human retroviruses.

[17]  U Landegren,et al.  A ligase-mediated gene detection technique. , 1988, Science.

[18]  D. Katzenstein,et al.  A mutation in human immunodeficiency virus reverse transcriptase and decline in CD4 lymphocyte numbers in long-term zidovudine recipients. , 1993, The Journal of infectious diseases.

[19]  Douglas D. Richman,et al.  The safety and efficacy of zidovudine (AZT) in the treatment of subjects with mildly symptomatic human immunodeficiency virus type 1 (HIV) infection a double-blind, placebo-controlled trial , 1990 .

[20]  V. Johnson,et al.  Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group , 1993, Antimicrobial Agents and Chemotherapy.

[21]  S D Kemp,et al.  Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. , 1991, Science.

[22]  J. Zack,et al.  Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle , 1992, Journal of virology.

[23]  W. Guerra.  Zidovudine in asymptomatic human immunodeficiency virus infection. A controlled trial in persons with fewer than 500 CD4-positive cells per cubic millimeter , 1991 .

[24]  H. Lazarus,et al.  Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia , 1965, Cancer.

[25]  D. Richman,et al.  Use of self-sustained sequence replication amplification reaction to analyze and detect mutations in zidovudine-resistant human immunodeficiency virus. , 1991, The Journal of infectious diseases.

[26]  D. Richman,et al.  Prevalence and Clinical Significance of Zidovudine Resistance Mutations in Human Immunodeficiency Virus Isolated from Patients after Long-Term Zidovudine Treatment , 1995 .