Increased detection of the HIV-1 reverse transcriptase M184V mutation using mutation-specific minority assays in a UK surveillance study suggests evidence of unrecognized transmitted drug resistance*

Abstract

The aim of the study was to estimate the levels of transmitted drug resistance (TDR) in HIV-1 using very sensitive assays to detect minority drug-resistant populations. MethodsWe tested unlinked anonymous serum specimens from sexual health clinic attendees, who had not received an HIV diagnosis at the time of sampling, by both standard genotyping and using minority detection assays. ResultsBy standard genotyping, 21 of 165 specimens (12.7%) showed evidence of drug resistance, while, using a combination of standard genotyping and minority mutation assays targeting three commonly observed drug resistance mutations which cause high-level resistance to commonly prescribed first-line antiretroviral therapy (ART), this rose to 32 of 165 (19.4%). This increase of 45% in drug resistance levels [95% confidence interval (CI) 15.2-83.7%; P 5 0.002] was statistically significant. Almost all of this increase was accounted for by additional detections of the M184V mutation. ConclusionsFuture surveillance studies of TDR in the United Kingdom should consider combining standard genotyping and minority-specific assays to provide more accurate estimates, particularly when using specimens collected from chronic HIV infections in which TDR variants may have declined to low levels.Keywords: drug resistance, HIV-1, minority-specific polymerase chain reaction, surveillance IntroductionThe use of genotypic resistance testing to detect drugresistant HIV type 1 variants has helped to guide decision making about appropriate antiretroviral therapy (ART) choices. Following evidence of transmission of drug resistance [2], which may compromise response to firstline therapy [3], routine screening for transmitted drug resistance (TDR) at the time of new diagnosis has been implemented [4]. This allows optimized first-line treatment, as well as surveillance of transmitted resistance.The levels of HIV-transmitted resistance in the United Kingdom peaked in 2002 at about 12% but subsequently fell to around 7-8% by 2006 [5]. It has been previously noted that there is discordance in the spectrum of resistance-associated mutations observed at transmission, compared with those emerging during ART in treated individuals [6,7]. The key lamivudine mutation, M184V, which is the most commonly observed mutation in treated patients, is seldom seen in viruses from untreated patients, DOI: 10.1111/j.1468-1293.2010.00882.x HIV Medicine (2011 r 2010 British HIV Association 250including those who have other drug resistance-associated mutations. This has been thought to be attributable either to it causing reduced transmissibility of the virus or to the rapid loss of this mutation in the absence of treatment as a result of its impact on viral fitness [6]. Standard resistance testing on plasma is limited to detecting viruses present as majority populations (420%) within the viral quasispecies. By contrast, some variants may only be present in low proportions, and thus avoid detection. A number of studies in ART-naïve patients have identified drug-resistant variants...

Prevalence of Drug-Resistant HIV Type 1 at the Time of Initiation of Antiretroviral Therapy in Portland, Oregon

“…It has been demonstrated that newer, more sensitive resistance testing assays in development can now detect mutations down to < 1%. [34][35][36][37][38][39] Therefore, we are potentially missing undetected mutations that may impact response to treatment. Finally, there was wide variability in the time between diagnosis and genotype testing in our patient population.…”

Subunit-Selective Mutational Analysis and Tissue Culture Evaluations of the Interactions of the E138K and M184I Mutations in HIV-1 Reverse Transcriptase

“…Under selection pressure with 3TC or FTC, both in vitro and in vivo, the M184I mutation usually emerges first and is rapidly replaced by the M184V mutation due to the relative fitness advantage of the latter over the former mutation (13,15,29,48). However, M184I/V mutations are rarely found in cases of transmitted drug resistance (TDR), although the M184V mutation can be detected in newly infected individuals with detection methods that are more sensitive than standard genotyping (12,47,52). As a consequence of low viral fitness, the prevalence of the M184V mutation as a single mutation in newly infected individuals wanes over time (52).…”

“…RT enzymes were expressed in Escherichia coli BL21 cells (Invitrogen, Burlington, Ontario, Canada) and purified by nickel affinity chromatography and Q Sepharose ion-exchange chromatography, as described previously (33). The polymerase activity of each recombinant RT preparation was evaluated in triplicate by using the synthetic homopolymeric poly(rA)/p(dT) [12][13][14][15][16][17][18] template/primer (T/P) (Midland Certified Reagent Company) as described previously (41). An active unit of RT was defined as the amount of enzyme that incorporates 1 pmol of dTTP in 10 min at 37°C.…”

“…Kinetics studies were carried out by a modification of a previously described method using homopolymeric poly(rA)/p(dT) [12][13][14][15][16][17][18] and complementary dTTP as the nucleotide substrate (54). The reaction mixture (10 l) contained 50 mM Tris-HCl (pH 7.8), 60 mM KCl, 6 mM MgCl 2 , 5 mM dithiothreitol (DTT), 0.5 U/ml poly(rA)/p(dT) [12][13][14][15][16][17][18] , RT enzymes, variable concentrations of the tracer [ 3 H]dTTP, and cold dTTP (0.2 to 200 M). Reactions were run at 37°C and quenched by the addition of 0.2 ml of 10% cold trichloroacetic acid and 20 mM sodium pyrophosphate to the mixtures; products were filtered onto Millipore 96-well MutiScreen HTS FC filter plates (catalog number MSFCN6B) and sequentially washed with 200 l of 10% TCA and 150 l of 95% ethanol.…”

“…1B). The activities of all of the RTs were determined by measuring dTTP incorporation using the poly(rA)/p(dT) [12][13][14][15][16][17][18] template/primer. In the RNA-dependent DNA polymerase reactions that measured rates of polymerization, the 497-nt HIV-1 PBS RNA template was used.…”