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

Monitoring for lamivudine (3TC) resistance is important both for the clinic al management of human immunodeficiency virus type 1 (HIV-1)-infected patie nts 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 triphosp hate (3TC-TP) in plasma, RT detection was done by the Amp-RT assay, an ultr asensitive PCR-based RT assay. Under our assay conditions, we found that 5 mu M 3TC TP inhibited RT activity from wild-type (WT), zidovudine-resistant , or nevirapine-resistant HIV-1 but not from HIV-1 carrying either the M184 V 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 sampl es, HIV-1 RT in 30 plasma specimens collected from 15 patients before and d uring therapy with 3TC was tested for evidence of phenotypic resistance by the;Imp-RT assay. The results were compared with those of genotypic analysi s, The RT in 12 samples was found to be 3TC sensitive, while the RT in 18 s amples had evidence of phenotypic resistance. All 12 samples with 3TC-sensi tive RT had WT genotypes at codon 185 and were retrieved before treatment w ith 3TC. In contrast, all 18 specimens with 3TC-resistant RT were postthera py samples. This assay provides a simple, rapid, and reliable method for th e detection of phenotypic resistance of HIV-1 to 3TC in plasma.