A Rapid Non-Culture-Based Assay for Clinical Monitoring of Phenotypic Resistance of Human Immunodeficiency Virus Type 1 to Lamivudine (3TC)

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 μM 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.

Measuring Enzymatic HIV-1 Susceptibility to Two Reverse Transcriptase Inhibitors as a Rapid and Simple Approach to HIV-1 Drug-Resistance Testing

“…Thus, simpler and less expensive drug resistance technologies may improve patient management. Previously explored technology relied on detecting and phenotyping HIV-associated RT activity in plasma by ultrasensitive biochemical assays [9], [17], [23], [24]. In this study we describe an improved biochemical approach to resistance testing for NNRTI and 3TC/FTC.…”

“…In this study we describe an improved biochemical approach to resistance testing for NNRTI and 3TC/FTC. The principle is based on our previously reported Amp-RT assays [9], [17] but has been improved in two major ways. First, we include real-time PCR for the detection of Amp-RT product which eliminates the need of ELISA quantitation of the PCR products, thus reducing time and cost.…”

“…As a competitive inhibitor, 3TC-TP inhibits RT activity more efficiently when dCTP concentration is reduced resulting in less 3TC-TP in the assay. We have previously shown by titrations of dCTP and 3TC-TP that 10 µM of dCTP was optimal for detecting both RT activity and its inhibition by 3TC-TP [17]. The RT reaction was carried out for 2 h at 37°C, followed by a 5 min inactivation of HIV-1 RT at 95°C.…”

“…Like other RT assays that are broadly reactive on all retroviruses, Amp-RT can detect generically RT activity from diverse retrovirus groups including HIV-1 non-subtype B and HIV-2 [15], [16]. We have previously demonstrated that this technology can also be used for HIV drug-resistance testing and have shown that biochemical detection of NNRTI and 3TC resistance by this method correlates well with genotypic and replication-based phenotypic assays [9], [17]. Here, we describe the development of improved Amp-RT assays that use real-time PCR detection and simplified virus capture from plasma.…”

K70Q Adds High-Level Tenofovir Resistance to “Q151M Complex” HIV Reverse Transcriptase through the Enhanced Discrimination Mechanism

“…Q151M by itself causes intermediate- to high-level resistance to zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), and low level resistance to abacavir (ABC) [15], [16], [17] without reducing viral fitness [18], [19]. Addition of the four associated mutations increases replication capacity of RT and results in high-level resistance to AZT, ddI, ddC, and d4T, 5-fold resistance to ABC and low-level resistance to lamivudine (3TC) and emtricitabine (FTC) [17], [18], [19], [20], [21]. Miller et al and Smith et al reported a 1.8-fold and 3.6-fold increase in resistance to tenofovir (TFV), respectively [22], [23].…”