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. 1999 Feb;43(2):264-70.
doi: 10.1128/AAC.43.2.264.

A rapid non-culture-based assay for clinical monitoring of phenotypic resistance of human immunodeficiency virus type 1 to lamivudine (3TC)

J García Lerma  1 , R F Schinazi, A S Juodawlkis, V Soriano, Y Lin, K Tatti, D Rimland, T M Folks, W Heneine
Affiliations

A rapid non-culture-based assay for clinical monitoring of phenotypic resistance of human immunodeficiency virus type 1 to lamivudine (3TC)

J García Lerma et al. Antimicrob Agents Chemother. 1999 Feb.

Abstract

Monitoring for lamivudine (3TC) resistance is important both for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with 3TC and for surveillance of transmission of 3TC-resistant HIV-1. We developed a novel non-culture-based assay for the rapid analysis of phenotypic resistance to 3TC of HIV-1 in plasma. The assay measures the susceptibility of HIV-1 reverse transcriptase (RT) activity to 3TC triphosphate (3TC-TP) in plasma. RT detection was done by the Amp-RT assay, an ultrasensitive PCR-based RT assay. Under our assay conditions, we found that 5 microM 3TC-TP inhibited RT activity from wild-type (WT), zidovudine-resistant, or nevirapine-resistant HIV-1 but not from HIV-1 carrying either the M184V mutation or multidrug (MD) resistance mutations (77L/116Y/151M or 62V/75I/77L/116Y/151M). Mixing experiments showed a detection threshold of 10% 3TC-resistant virus (M184V) in a background of WT HIV-1. To validate the assay for the detection of phenotypic resistance of HIV-1 to 3TC in plasma samples, HIV-1 RT in 30 plasma specimens collected from 15 patients before and during therapy with 3TC was tested for evidence of phenotypic resistance by the Amp-RT assay. The results were compared with those of genotypic analysis. The RT in 12 samples was found to be 3TC sensitive, while the RT in 18 samples had evidence of phenotypic resistance. All 12 samples with 3TC-sensitive RT had WT genotypes at codon 184 and were retrieved before treatment with 3TC. In contrast, all 18 specimens with 3TC-resistant RT were posttherapy samples. This assay provides a simple, rapid, and reliable method for the detection of phenotypic resistance of HIV-1 to 3TC in plasma.

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Figures

FIG. 1
FIG. 1
Detection of phenotypic resistance to 3TC by measuring levels of RT inhibition by the Amp-RT assay. (A) Inhibition of 10−7 U of RT activity from WT (xxBRUpitt) and 3TC-resistant (M184Vpitt) HIV-1 by different concentrations of 3TC-TP. (B) Inhibition of RT activity (from 10−6 to 10−8 U) from WT (xxBRUpitt) and 3TC-resistant (M184Vpitt) HIV-1 by 5 μM 3TC-TP.
FIG. 2
FIG. 2
Analysis of the specificity of the Amp-RT-based phenotypic assay for detection of 3TC resistance by testing RTs from several HIV-1 reference clones in the presence and absence of 5 μM 3TC-TP. Lane 1, WT clone xxBRUpitt; lane 2, 3TC-resistant clone M184Vpitt; lane 3, nevirapine-resistant clone 181C/YEU; lane 4, 3TC- and nevirapine-resistant clone M184V/Y181CEU; lane 5, AZT-resistant clone HIV-1RTMC/MT-2; lane 6, water control (Neg, negative).
FIG. 3
FIG. 3
Evaluation of the detection threshold of 3TC resistance by the Amp-RT assay and comparison with the genotypic analysis at codon 184 by the HIV-1 LiPA. (A) Levels of RT inhibition by 3TC-TP (5 μM) in mixtures of WT clone xxBRUpitt and 3TC-resistant HIV-1 clone M184Vpitt. (B) Genotypic detection of the M184V mutation in the same mixtures by the HIV-1 LiPA. Conj. control, conjugate control.
FIG. 4
FIG. 4
Determination by the Amp-RT assay of the IC50s and IC90s of 3TC-TP for HIV-1 RT carrying MD resistance mutations in the presence of increasing concentrations of 3TC-TP. HIV-1SUM9 and xxBRUpitt are WT; Y181CEU is nevirapine resistant, M184Vpitt is 3TC resistant, and HIV-1SUM8, HIV-1SUM12, and HIV-1SUM13 are MD-resistant HIV-1 clones.
FIG. 5
FIG. 5
Detection of phenotypic resistance to 3TC of HIV-1 in plasma by the Amp-RT assay. Results of duplicate tests with plasma samples from three HIV-1-infected patients (patients 6, 8, and 14) obtained at different time points before therapy (basal and pre-therapy) and during antiretroviral therapy (1 to 44 weeks) with 3TC are shown. SC, HIV-1-, HIV-2-, HTLV-I-, and HTLV-II-seronegative control; w, water control.
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