Nonisotopic hybridization assay for determination of relative amounts of genotypic human immunodeficiency virus type 1 zidovudine resistance

Eastman, Peter; Boyer, E. W.; Molé, L; Kolberg, Janice A.; Urdea, Mickey S.; Holodniy, Mark

doi:10.1128/jcm.33.10.2777-2780.1995

Cited by 20 publications

(17 citation statements)

References 15 publications

(24 reference statements)

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“…Although sequencing provides precise information about the nucleotide changes, the procedure involves a heavy commitment of time and labor, and the information obtained describes only the predominant viral populations. Techniques such as nucleotide-specific probe hybridizations (10,12,21,25,40) and sequencing of uncloned PCR products (16,46,47) have been specifically designed to detect the true in vivo existence of a large number of viral quasispecies. The screening assay described here offers a simple procedure for identifying amino acid substitutions at codons 82 and 90 of the protease gene in uncloned viral species.…”

Section: Discussionmentioning

confidence: 99%

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Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection

Vasudevachari

Zhang

Imamichi

et al. 1996

Antimicrob Agents Chemother

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Patient human immunodeficiency virus type 1 (HIV-1) isolates that are resistant to protease inhibitors may contain amino acid substitutions L10I/V, M46L/I, G-48V, L63P, V82A/F/T, I84V, and L90M in the protease gene. Substitutions at positions 82 and/or 90 occur in variants that display high levels of resistance to certain protease inhibitors. Nucleotide substitutions at these two sites also lead to the loss of two HindII restriction enzyme digestion sites, and these changes make possible a rapid procedure for the detection of drug-resistant variants in patients on protease inhibitor therapy. This procedure was used to detect the emergence of mutated viruses at various times after the initiation of therapy with the HIV-1 protease inhibitor indinavir. The method includes viral RNA isolation from plasma and reverse transcription PCR amplification of the protease gene with fluorescence-tagged primers. The PCR product is digested with HindII, the cleavage products are separated on a urea-acrylamide gel in a DNA sequencer, and the extent of cleavage is automatically analyzed with commercially available software. In viruses from 34 blood samples from four patients, mutations leading to an amino acid change at residue 82 appeared as early as 6 weeks after the start of therapy and persisted throughout the course of the study period (48 weeks). Mutations leading to double substitutions at residues 82 and 90 were seen at a lower frequency and appeared later than the change at position 82. The changes detected by restriction enzyme cleavage were confirmed by DNA sequencing of the cloned protease genes by reverse transcription PCR amplification of viral RNA from isolates in plasma. In addition to the changes at positions 82 and 90, we have identified M46L/I, G48V, and I54V substitutions in isolates derived from indinavir-treated patients. HindII analysis of uncloned, PCR-amplified DNA offers a rapid screening procedure for the detection of virus isolates containing mutations at amino acid residues 82 and 90 in the HIV-1 protease gene. By using other restriction enzymes, the same method can be used to detect additional protease drug-resistant variants and is generally applicable for the detection of mutations.

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Section: Discussionmentioning

confidence: 99%

“…(B) HindII analysis of fluorescence-labeled PCR products. Lanes 1 through 12, sequential plasma samples obtained from patient H before drug administration and at3,6,10,16,20,24,27,32,36,40, and 44 weeks of indinavir administration. The sizes of the denatured fragments of the internal lane standards are given on the left side (in base pairs).…”

mentioning

confidence: 99%

Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection

Vasudevachari

Zhang

Imamichi

et al. 1996

Antimicrob Agents Chemother

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“…The syncytium-inducing capacities of the viral isolates were measured in MT-2 cells by a standard methodology (15). Genotypic analysis of amino acid position 215 of the RT gene was measured in the viral isolates as reported previously (11).…”

Section: Methodsmentioning

confidence: 99%

Randomized, controlled phase I/II, trial of combination therapy with delavirdine (U-90152S) and conventional nucleosides in human immunodeficiency virus type 1-infected patients

Davey

Chaitt

Reed

et al. 1996

Antimicrob Agents Chemother

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Delavirdine mesylate (DLV) is a potent nonnucleoside reverse transcriptase inhibitor with activity specific for human immunodeficiency virus type 1. In the present phase I/II study we evaluated the safety, toxicity, pharmacokinetics, and antiretroviral activities of two-drug and three-drug combinations of DLV and conventional doses of nucleoside analogs compared with those of both DLV monotherapy and two-drug nucleoside analog therapy. A total of 85 human immunodeficiency virus type 1 infected patients with CD4 counts of 100 to 300 cells per mm3 were enrolled in two periods: in the first period patients were randomized to receive either zidovudine (ZDV) plus didanosine (group 1) or ZDV plus didanosine plus escalating doses (400 to 1,200 mg/day) of DLV (group 2). In the second period, patients were randomized to receive either 1,200 mg of DLV alone per day (group 3) or ZDV plus 1,200 mg of DLV per day (group 4). DLV demonstrated good oral bioavailability at all five doses tested. The major toxicity was a transient mild rash which appeared in 44% of all DLV recipients. Overall, group 2 patients demonstrated more sustained improvements in CD4 counts, percent CD4 cells, branched DNA levels, p24 antigen levels, and virus titers in plasma than group 1, 3, or 4 patients. The magnitude of the response correlated with the intensity of prior nucleoside analog treatment, the non-syncytium-inducing or syncytium-inducing viral phenotype at baseline, and the presence of a wild-type codon at amino acid position 215 in the baseline reverse transcriptase genotype. Despite a transient rash, DLV therapy was well tolerated. Combination therapy with DLV and nucleoside analogs appears promising, with the three-drug combination appearing to be more potent that either two-drug combinations or monotherapy.

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“…Several assays have been developed to rapidly detect point mutations (3,5,6,9,12,13,15). In 1991, Larder et al developed a nested PCR protocol for detecting zidovudine resistance mutations using oligonucleotides specific for either wildtype or mutant HIV-1 RT DNA (13).…”

mentioning

confidence: 99%

“…Modifications of PCR, such as using a hot start and other heat-stable polymerases, should further improve the sensitivity and specificity of sequence-specific PCR (11,21). In addition, some point mutation assays are able to quantify the proportions of wild-type and mutant sequences within a mixture (3,8,9). Because point mutation assays are being used to study the timing and significance of drug resistance during antiretroviral therapy (2,7,8,(17)(18)(19), it has become important to determine the accuracy and reproducibility of these assays.…”

mentioning

confidence: 99%

Interlaboratory comparison of sequence-specific PCR and ligase detection reaction to detect a human immunodeficiency virus type 1 drug resistance mutation. The AIDS Clinical Trials Group Virology Committee Drug Resistance Working Group

et al. 1996

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Sequence-specific PCR was used in six laboratories and a ligase detection reaction was used in one laboratory to detect the zidovudine-resistance mutation at codon 215 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase DNA. The genotypes of 27 different clinical samples, including cultured HIV-1 isolates, peripheral blood mononuclear cells, and plasma, were correctly identified by 140 of 154 (91%) assays.

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Nonisotopic hybridization assay for determination of relative amounts of genotypic human immunodeficiency virus type 1 zidovudine resistance

Cited by 20 publications

References 15 publications

Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection

Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection

Randomized, controlled phase I/II, trial of combination therapy with delavirdine (U-90152S) and conventional nucleosides in human immunodeficiency virus type 1-infected patients

Interlaboratory comparison of sequence-specific PCR and ligase detection reaction to detect a human immunodeficiency virus type 1 drug resistance mutation. The AIDS Clinical Trials Group Virology Committee Drug Resistance Working Group

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