Thymidine-associated Mutations Research Articles

Withdrawing inactive NRTIs in HIV-1 subjects with suppressed viraemia: a randomized trial.

Extensively pretreated subjects with virological failure (VF) may receive salvage regimens containing NRTIs with only residual or no activity. Once virological suppression is achieved, their contribution remains elusive. This was a multicentre, randomized, prospective study. Subjects with at least one prior VF, HIV-1 RNA <50 copies/mL for ≥6 months and receiving a regimen with at least two active drugs (one of them a boosted PI) were randomized 1:1 to stop (experimental arm) or maintain (control arm) NRTIs. EudraCT: 2012-000198-21. Ninety subjects were randomized (experimental, n = 45; and control, n = 45). The mean age was 50 years, 80% were male, the mean CD4+ cell count was 542 cells/mm(3) and the median number of prior VFs was 3. Seventy-four subjects (82%) harboured the mutation M184V/I and the median number of thymidine-associated mutations was 3 (IQR: 0-4). In the experimental arm, thirty-two (71%) subjects removed one NRTI and 13 (29%) subjects removed two. Twenty-two of 45 (49%) discontinued tenofovir disoproxil fumarate. Forty-one of 45 (91.1%, experimental arm) and 44 of 45 (97.8%, control arm) had HIV-1 RNA <50 copies/mL at 48 weeks (difference: -6.7%; 95% CI: -17.4, 4.1). In a post-hoc analysis allowing NRTI reintroduction, efficacy rates were 95.6% and 97.8%, respectively (difference: -2.2%; 95% CI: -7.2, 2.7). Rates of discontinuation at 48 weeks were 2% in both arms. One subject developed a late VF with resistance selection. In patients receiving a successful multidrug salvage regimen with at least two active drugs (one a boosted PI), the withdrawal of inactive NRTIs was safe, rates of VF were low and drug resistance was uncommon at 48 weeks in this small study. This strategy could potentially prevent long-term toxicities, reduce the number of drugs and reduce costs if non-inferiority was met in a fully powered trial.

HIV-1 Reverse Transcriptase (RT) Polymorphism 172K Suppresses the Effect of Clinically Relevant Drug Resistance Mutations to Both Nucleoside and Non-nucleoside RT Inhibitors

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT(172K)) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the k(cat)/K(m) values for dNTP. Surface plasmon resonance experiments revealed that RT(172K) decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT(172K) results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 Å) crystal structure of RT mutated at 172 and compared crystal structures of RT(172R) and RT(172K) bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between α-helix E (where 172 resides) and the active site β9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.

A pyrophosphatase activity associated with purified HIV-1 particles

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.

K65R in Subtype C HIV-1 Isolates from Patients Failing on a First-Line Regimen Including d4T or AZT: Comparison of Sanger and UDP Sequencing Data

BackgroundWe and others have shown that subtype C HIV-1 isolates from patients failing on a regimen containing stavudine (d4T) or zidovudine (AZT) exhibit thymidine-associated mutations (TAMs) and K65R which can impair the efficacy of Tenofovir (TDF) at second line. Depending on the various studies, the prevalence of K65R substitution as determined by the Sanger method ranges from 4 to 30%. Our aim was to determine whether ultra-deep pyrosequencing (UDPS) could provide more information than the Sanger method about selection of K65R in this population of patients.Methods27 subtype C HIV-1 isolates from treated patients failing on a regimen with d4T or AZT plus lamivudine (3TC) plus nevirapine (NVP) or efavirenz (EFV) and who had been sequenced by Sanger were investigated by UDPS at codon 65 of the reverse transcriptase (RT). 18 isolates from naïve patients and dilutions of a control K65R plasmid were analysed by Sanger plus UDPS.ResultsAnalysis of Sanger sequences of subtype C HIV-1 isolates from naïve patients exhibited expected polymorphic substitutions compared to subtype B but no drug resistance mutations (DRMs). Quantitation of K65R variants by UDPS ranged from <0.4% to 3.08%. Sanger sequences of viral isolates from patients at failure of d4T or AZT plus 3TC plus NVP or EFV showed numerous DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) including M184V, thymidine-associated mutations (TAMs) plus DRMs to non- nucleoside reverse transcriptase inhibitors (NNRTIs). Two K65R were observed by Sanger in this series of 27 samples with UDPS percentages of 27 and 87%. Other samples without K65R by Sanger exhibited quantities of K65R variants ranging from <0.4% to 0.80%, which were below the values observed in isolates from naïve patients.ConclusionsWhile Sanger sequencing of subtype C isolates from treated patients at failure of d4T or AZT plus 3TC plus NVP or EFV exhibited numerous mutations including TAMs and 8% K65R, UDPS quantitation of K65R variants in the same series did not provide any more information than Sanger.

HIV-1 Polymerase Inhibition by Nucleoside Analogs: Cellular- and Kinetic Parameters of Efficacy, Susceptibility and Resistance Selection

Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.

Foscarnet salvage therapy efficacy is associated with the presence of thymidine-associated mutations (TAMs) in HIV-infected patients

Background Salvage therapy based on foscarnet plus a thymidine analog is effective in patients with advanced-stage HIV disease and viruses harbouring multiple drug-resistance mutations. Objective To identify viral genetic determinants associated with the virological efficacy of foscarnet salvage therapy. Study design Thirteen patients received foscarnet at a fixed dose of 80 mg/kg twice daily for 14 days, in combination with zidovudine or stavudine. Results The baseline median HIV viral load and CD4 cell count were 5.10 log 10 copies/ml and 23 cells/mm 3, respectively. Following foscarnet therapy, viral load fell by a median of 1.84 log 10 copies/ml (range: −0.29 to −2.82), and by at least 1 log 10 copies/ml in 11 patients, all of whom harboured viruses with at least three thymidine-associated mutations (TAMs). The two patients with smaller declines in viral load (<0.50 log 10 copies/ml) harboured viruses with only one or zero TAMs. Conclusions These findings corroborate, in vivo, the impact of TAMs on HIV susceptibility to foscarnet. The virological response to foscarnet salvage therapy in multiclass-experienced patients may thus differ according to the number of TAMs.

Phylogenetic and Genetic Analysis of Feline Immunodeficiency Virus gag , pol , and env Genes from Domestic Cats Undergoing Nucleoside Reverse Transcriptase Inhibitor Treatment or Treatment-Naïve Cats in Rio de Janeiro, Brazil

Feline immunodeficiency virus (FIV) is the Lentivirus responsible for an immunodeficiency-like disease in domestic cats (Felis catus). FIV is divided into five phylogenetic subtypes (A, B, C, D, and E), based on genetic diversity. Knowledge of the geographical distribution of subtypes is relevant for understanding different disease progressions and for vaccine development. In this study, viral sequences of 26 infected cats from Rio de Janeiro, 8 undergoing treatment with zidovudine (AZT) for at least 5 years, were successfully amplified from blood specimens. gag capsid (CA), pol reverse transcriptase (RT), and env gp120 (V3-V4) regions were analyzed to determine subtypes and to evaluate potential mutations related to antiretroviral drug resistance among treated cats. Subtyping based on phylogenetic analysis was performed by the neighbor-joining and maximum likelihood methods. All of the sequences clustered with subtype B in the three regions, exhibiting low genetic variability. Additionally, we found evidence that the same virus is circulating in animals in close contact. The analysis of FIV RT sequences identified two new putative mutations related to drug resistance located in the RT "finger" domain, which has 60% identity to human immunodeficiency virus (HIV) sequence. Amino acid change K-->R at codons 64 and 69 was found in 25% and 37.5% of the treated animals, respectively. These signatures were comparable to K65R and K70R thymidine-associated mutations found in the HIV-1 HXB2 counterpart. This finding strongly suggests a position correlation between the mutations found in FIV and the K65R and K70R substitutions from drug-resistant HIV-1 strains.

The use of β-D-2,6-diaminopurine dioxolane with or without mycophenolate mofetil in drug-resistant HIV infection

We evaluated the safety, tolerability and antiretroviral activity of beta-D-2,6-diaminopurine dioxolane (DAPD; amdoxovir) with or without mycophenolate mofetil (MMF) in HIV-1 infection following extensive antiretroviral therapy (ART). Oral DAPD 500 mg twice daily with placebo or MMF 500 mg twice daily was added to failing ART. HIV-1 RNA viral load (VL) decline to week 2 was analyzed by intent-to-treat, using rank-based tests. Patients with VL decline > 0.5 log10 copies/ml at week 2 (virologic response, VR) optimized ART and continued therapy for up to 96 weeks. Forty adults with median VL 4.5 log10 copies/ml, median 184 CD4+ cells/microl, and a median of 6 nucleoside reverse transcriptase inhibitor (NRTI) mutations (range, 1-8) were randomized. Median VL reduction at week 2 was -0.26 log10 copies/ml (P < 0.0001). Response to DAPD/placebo (median -0.37 log10 copies/ml) was unexpectedly greater than to DAPD/MMF (median -0.23 log10 copies/ml), although this difference was not statistically significant (P = 0.59). MMF appeared to lower concentrations of DAPD and its metabolite dioxolane guanosine. Of 10 patients with VR (DAPD 7, DAPD/MMF 3), four persisted beyond week 24. VR was more frequent with < or = 5 baseline NRTI mutations (P = 0.12) or < 4 thymidine-associated mutations (TAMs) without E44D or V118I (P = 0.08). Twenty-three patients received extended DAPD +/- MMF; five beyond week 24. Few adverse events were related to study medications. The addition of DAPD +/- MMF to failing therapy appears safe and well tolerated. DAPD had significant activity at week 2 (mean -0.35 log10) in heavily pretreated patients that was not augmented by MMF.

Effects of the Translocation Status of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on the Efficiency of Excision of Tenofovir

The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can occur only when the reverse transcriptase complex exists in its pretranslocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the posttranslocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiencies of the reaction in response to changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as those seen with 3'-azido-3'-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to the case for AZT-MP, the removal of tenofovir was highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pretranslocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex posttranslocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the posttranslocational state and by dead-end complex formation, which provides a degree of protection from excision in the cellular environment.

Antiviral activity of low‐dose alovudine in antiretroviral‐experienced patients: results from a 4‐week randomized, double‐blind, placebo‐controlled dose‐ranging trial*

Alovudine inhibits replication of highly nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV strains in vitro. However, dose-dependent safety concerns resulted in its initial development being halted. Recently, a 4-week course of alovudine 7.5 mg/day added to a stavudine-free failing regimen yielded a significant decrease in viral load by -1.88 log(10) HIV-1 RNA copies/mL. The magnitude of the reduction in viral load suggested that lower doses might still be effective while offering adequate safety during long-term use. To determine whether lower dosages of alovudine still provide significant antiviral activity in patients with broad NRTI resistance. A randomized, double-blind, placebo-controlled trial investigating three doses of alovudine (0.5, 1 and 2 mg) or placebo added for 4 weeks to a failing regimen in patients with evidence of NRTI-resistant HIV strains [>or=2 thymidine-associated mutations (TAMs)]. The primary endpoint was the mean viral load reduction between baseline and week 4. Seventy-two patients were enrolled in the study: 21, 13, 18 and 20 in the placebo and 0.5, 1 and 2 mg arms, respectively. Baseline median CD4 count and viral load were 298 cells/microL (range 44-692 cells/microL) and 3.9 log(10) copies/mL (range 2.5-5.2 log(10) copies/mL), respectively. Baseline viral isolates harboured a median of four TAMs. Alovudine was added to a median four-drug failing regimen. At week 4, compared with placebo, mean viral load changes were -0.42 log(10) [95% confidence interval (CI) -0.67 to -0.18] and -0.30 log(10) (-0.55 to -0.06) in the 2 and 1 mg arms, respectively. There was no significant change in CD4 cell count. Alovudine was well tolerated. A 4-week course of alovudine 2 mg/day provided a modest but significant viral load reduction in patients harbouring viruses with a median of four TAMs.

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre- and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3' end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.

Didanosine Enteric-Coated Capsule

Didanosine, which is a synthetic nucleoside analogue intracellularly phosphorylated to the active metabolite, inhibits the activity of HIV-1 reverse transcriptase by competing with the natural substrate. Currently, didanosine is mainly provided as an enteric-coated capsule. In vitro, the molecule is active against laboratory strains and clinical isolates of HIV-1 in resting and activated T cells and monocyte/macrophages. Didanosine may select for resistance mutations that may render the drug inactive against the virus; L74V and K65R remain as the main didanosine-related mutations. In vitro, phenotypic susceptibility to didanosine was decreased beyond a defined fold change clinical cut-off (1.7), and it is considered that genotypic resistance exists when five thymidine-associated mutations or four plus M184V are present. In vivo, clinical studies have shown that didanosine retains significant antiviral activity in patients who have up to five nucleoside analogue mutations at baseline. Didanosine is useful in patients with no previous therapy, as well as in experienced patients in whom one or more antiretroviral regimens has failed.Enteric-coated didanosine is taken once daily, its co-administration with food has been recently evaluated and a reduction of the efficacy of the antiretroviral treatment was not observed. Administered with lamivudine (or emtricitabine), it can be considered a good alternative for use in the nucleoside analogue backbone included in combination therapies for antiretroviral-naive patients. Didanosine could be used in initial treatments for patients intolerant of zidovudine, abacavir or tenofovir. It can be included in once-daily combination regimens, which are more convenient and patient friendly.Prospective, observational and open-label studies, as well as clinical trials (with durations between 24 and 96 weeks), have demonstrated the safety and efficacy of didanosine plus lamivudine (or emtricitabine) plus efavirenz (or nevirapine) in previously untreated HIV-1-infected patients. The administration of didanosine to treatment-experienced patients has been evaluated in two different contexts: patients in whom previous therapies have failed (rescue therapy) and those with controlled viraemia who are switched to a didanosine-containing regimen for simplification.Adverse events associated with the administration of didanosine have been well known since the initial clinical trials with the drug. Gastrointestinal intolerance, peripheral neuropathy and hyperamylasaemia/pancreatitis were the most frequently reported. In the highly active antiretroviral therapy (HAART) era, the rate of adverse events has decreased. The tolerability of didanosine has been clearly improved with the development of the enteric-coated capsule. Severe manifestations of mitochondrial toxicity, including lactic acidosis and abnormal fat distribution, are rare complications, and occur most frequently when didanosine is used in combination with stavudine.

Efficacy and safety of tenofovir double‐dose in treatment‐experienced HIV‐infected patients: The tenoplus study

Drug resistance is an increasing problem in the treatment of HIV infection. Tenofovir has been shown to inhibit HIV replication even with thymidine-associated resistance mutations (TAMs) if they are limited to two or less. Double-dose of tenofovir disoproxil fumarate (TDF) (600 mg QD) was used to determine weather the drug could be virologically effective in patients harbouring HIV-strains resistant to nucleoside analogues (NRTI). A pilot, open, non-comparative add-on study, where patients failing a current antiretroviral regimen, with at least two TAMs, and naive for tenofovir, were given tenofovir 600 mg once-daily for 4 weeks, in addition to their current failing antiretroviral regimen. The primary end-point was the percentage of patients with plasma viral load (VL) reduction of at least 0.8 log(10) between baseline and week 4 (W4). Ten patients were enrolled. At baseline, the median viral load was 3.66 log(10) copies/ml (range 3.13-4.03) and the median CD4 cell count was 407/mm(3) (range 136-1102). The percentage of patients with reduction the viral load > or =0.8 log(10) was 40% at W4. After 4 weeks of treatment with tenofovir 600 mg, the median decrease in the viral load was -0.61 log(10) (range -0.05; -0.88) and the median gain of CD4 was +109/mm(3). Despite a twofold increase tenofovir plasma concentrations, no serious drug-related adverse event were recorded except for one patient experiencing an de Fanconi syndrome at week 2. This add-on pilot study supports the concept of double dose tenofovir to virologically overcome the decreased sensitivity of NRTI-resistant viruses. However, the safety of this regimen needs to be considered carefully.

Rate of Virologic Failure and Selection of Drug Resistance Mutations Using Different Triple Nucleos(t)ide Analogue Combinations in HIV-Infected Patients

Virologic failure seems to occur more frequently in HIV-infected patients treated with triple nucleoside analogue (NA) combinations than with regimens including nonnucleoside reverse transcriptase inhibitors or protease inhibitors. However, the rate of failure and resistance profiles may differ with distinct triple NA combinations. A retrospective review of all HIV-infected individuals who received triple NA combinations at our institution was conducted. Virologic failure was defined as lack of achievement of plasma HIV-RNA<50 copies/ml at week 16 following initiation of antiretroviral therapy or as viral rebound in subjects with prior undetectable viremia. Genotypic analyses were performed at the time of first virological failure. Of the 261 patients identified, 13 were drug naive, 126 had underwent simplification, and 122 were antiretroviral-experienced patients with detectable viral load. Virologic failure was recorded in 95 (36.4%) after an average follow-up of 19 months. Rates were 0.67 in drug-naive, 0.55 in simplification, and 2.38 in rescue interventions for 100 persons-month follow-up. Factors associated with virologic failure in the multivariate Cox regression analysis were rescue vs naive or simplification strategies (OR 2.6; 95% CI 1.6-4.2) and using tenofovir as part of the combination (OR 2.04; 95% CI 1.3-3.2). In contrast, the use of AZT prevented virologic failure (OR 0.52; 95% CI 0.3-0.8). M184V was the most frequent resistance mutation (75.4%), followed by T215Y (52.5%) and K65R (14.8%). Of note, K65R did not develop in patients taking AZT nor in those with prior thymidine-associated mutations (TAMs). Conversely, subjects who developed K65R did not accumulate TAMs. Virologic failure is relatively frequent in patients treated only with triple NA regimens, particularly in the setting of rescue therapy. The use of TDF might be associated with a higher risk of virologic failure and, conversely, AZT might be protective. The presence of TAMs precluded the selection of K65R in patients treated with TDF. Resistance pathways for TDF and thymidine analogues seem to be divergent and could be the basis to explore a synergism between these drugs.

Antiretroviral activity of didanosine in patients with different clusters of reverse transcriptase mutations

Patterns of mutations associated with didanosine (ddI) resistance are still a controversial issue. The correlation between different clusters of reverse transcriptase mutations with the short-term virological activity of ddI when added to a failing regimen was examined in 40 patients. The median fall in plasma viral load at week 4 was 0.67 log10 copies/ml. There was good correlation between the median fall in plasma HIV RNA levels and the number of nucleoside-associated (P = 0.0152) or thymidine-associated (P = 0.0142) mutations. In conclusion, ddI retained substantial antiretroviral activity when the number of nucleoside-associated or thymidine-associated mutations was less than four.

Virologic and Immunologic Efficacy of the Tenofovir/Didanosine/Lamivudine Regimen

To the Editor: Antiretroviral regimens combining the nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir (TDF), lamivudine (3TC), and abacavir or didanosine (ddI) have recently been linked to unexpectedly high rates of virologic failure, raising serious concerns about NRTI-only strategies. The European Agency for Evaluation of Medicinal Products recommended that the TDF/ddI/3TC combination should not be used, particularly as a once-a-day (OAD) regimen.1 This combination is frequently associated with the emergence of the K65R and M184V/I mutations.2 However, there is evidence that thymidine-associated mutations (TAMs) may protect against virologic failure and K65R selection during this treatment.3-6 The combination of TDF, 3TC, and abacavir or ddI therefore appears to provide a low genetic barrier to resistance, especially in previously untreated patients, as suggested by the results of in vitro studies and retrospective cohorts.7-11 Coadministration of TDF and a ddI dose of 400 mg/day appears to have a deleterious effect on the CD4 cell count, despite complete viral suppression. An imbalance in adenosine metabolites resulting from TDF inhibition of purine nucleoside phosphorylase in CD4 cells may explain this phenomenon.12-14 We reviewed the files of all consecutive patients who had been prescribed TDF 300 mg, didanosine 250 mg (reduced dose), and 3TC 300 mg daily, in an OAD schedule, between January 2002 and December 2004 in our center unit. Changes in the CD4 T-cell count and plasma viral load (pVL) were analyzed, together with the results of genotypic testing at baseline and during treatment. The patients were stratified by the baseline pVL (< or >500 copies/mL). Kaplan-Meier analysis was used to compare the outcomes in the 2 strata. Forty-two patients were enrolled, of whom 64% were men. The median age was 39.0 years (range, 24.7-58.2 years). The median duration of previous antiretroviral therapy was 6.3 years (range, 0-14.2 years), and the patients had received a median of 4 lines (range, 0-24 lines) of antiretroviral therapy. Three patients (7%) were antiretroviral-naive. The median duration of the OAD regimen was 16.8 months (range, 10.6-29.8 months). The median baseline CD4 cell count was 429.5 cells/mm3 (range, 9.3-1578 cells/mm3), and the baseline pVL was 3.25 log10 copies/mL (range, 1.7-5.7 log10 copies/mL). Genotyping showed that 31% of the patients had at least 3 TAMs at baseline. None of the antiretroviral-experienced patients developed the K65R mutation during the OAD regimen. In contrast, all 3 antiretroviral-naive patients developed both K65R and M184V mutations and had virologic failure. Overall, the slope of the CD4 cell count during the study period was -18.1/year (range, -594.3-1041.8/year). The median CD4 cell count did not differ significantly between baseline and the end of follow-up (P = 0.56). Overall, 52% of patients had a sustained pVL of less than 500 copies/mL (47.4% <50 copies/mL) after 96 weeks of follow-up. Nineteen patients had a baseline pVL of less than 500 copies/mL. Kaplan-Meier analysis showed a significant difference in pVL kinetics between the 2 strata (Fig. 1), whereas the CD4 cell count kinetics were similar (median, -29/year [range, -362-1042/year], P = 0.32 vs 13/year [range, -170-594], P = 0.47). Overall, the median CD4 cell count did not fall relative to baseline.FIGURE 1: Kaplan-Meier analysis: time on treatment (3TC-ddI-TDF).Experienced patients who switched to the OAD regimen when they already had undetectable plasma viremia continued to have a sustained virologic response, despite the presence of TAMs, such as T215Y, or the 3TC-associated mutation M184V at baseline. No resistance mutations specific for TDF were present at baseline in experienced patients. The results of this observational cohort study show the efficacy of the triple NRTI regimen consisting of TDF, ddI, and 3TC. The results are less worrisome than those of previous studies of triple NRTI regimens in naive patients and/or with ddI standard dosing. Indeed, low-dose ddI plus TDF and 3TC provided sustained virologic control and CD4 cell count stability. Romina Quercia, MD* Corinne Amiel, MD† Elisete Da Silva Marechal, MS* Marie-Gisèle Lebrette, MD* Sandrine Thevenet, CRA* Stéphanie Renaud, PD‡ Veéronique Schneider, MD† Ali Kara, MD† Sabine Guessant, DP‡ Gilles Pialoux, MD, PhD* *Infectious Disease Unit †Virology, ‡Pharmacy Tenon Hospital, Paris, France [email protected]

A case–control study of HIV patients with the K65R mutation in the reverse transcriptase gene with virological failure

The K65R mutation in the reverse transcriptase gene can be selected in vitro by tenofovir, as well as by zalcitabine, didanosine and abacavir [1–3]. The importance of this mutation lies in its possible role in multiresistance, which could severely hamper the chances of rescue in HIV patients treated with nucleoside analogues [4–6]. This mutation has also been detected over recent years in patients with early failure who started or simplified a combination treatment regimen with tenofovir as one of the three analogues [7–9]. Although the K65R mutation remains relatively uncommon [10], an increase in its prevalence has been observed over the past few years [11–14]. This trend probably results from the increased use of drugs that select for this mutation, such as tenofovir and didanosine [14]. We determined the incidence and prevalence of the K65R mutation in patients with virological failure and assessed the associated clinical, therapeutic and mutational characteristics, as well as the possibility of rescue. We undertook a genotype study (Trugene HIV-1 genotyping kit; Bayer Healthcare Diagnostics, Tarrytown, New York, USA) as part of a multicentre, 1: 1 case–control study including all patients with virological failure from 12 hospitals in southern Spain. Cases were defined as patients with the K65R mutation after virological failure, and controls consisted of patients matched for age (± 5 years), sex and time of failure (± 4 weeks) who did not have the K65R mutation. The study was undertaken from October 2001 to December 2004. Comparisons of quantitative data between the two groups were assessed using the Mann–Whitney test. Chi-squared statistics were used to test for associations between two qualitative variables, with Yates correction when necessary. All P values are two-tailed. Data were analysed using SPSS statistical software (SPSS Inc., Chicago, Illinois, USA). A total of 2 317 resistance tests were performed in patients whose antiretroviral therapy failed during the study period. The K65R mutation was identified in 64 isolates, representing a prevalence of 2.7%. The rate of the K65R mutation increased over time, from 2.2% in 2001 to 4.3% in 2004. The most common nucleoside reverse transcriptase inhibitors backbone in the antiretroviral regimen was didanosine–tenofovir and the most used drugs were didanosine, tenofovir and abacavir (Table 1). This was the first failure in only 14 cases. All patients except two were rescued with protease inhibitors (PI), with a good response in 72.7% (40/55). Failure with a PI regimen and the presence of thymidine-associated mutations (TAM) were more common in controls, and the use of didanosine, tenofovir and didanosine–tenofovir was more frequent in cases. The CD4 lymphocyte nadir and at failure were lower in cases than in controls. K65R was associated with M184V in 22 cases.Table 1: Patient characteristics with and without the K65R mutation.An increase has occurred in our area over the past few years in the incidence of the K65R mutation in patients with virological failure. The rates are similar to those of other European and American series [10,12,14], except for that of Valer et al. [13], who reported a higher incidence of 10%. As with other series, the mutation was associated with tenofovir and didanosine therapy, especially if they were used with another analogue or non-analogue. The mutation is much less common in association with the failure of PI therapy. The appearance of TAM somehow prevents the appearance of K65R, which reflects the divergent and antagonistic pathways of analogue resistance. Nevertheless, the appearance of this mutation does not imply a worse prognosis and rescue is possible in most patients, perhaps related to the worse viral fitness produced by this mutation, especially in association with M184V [10,15]. In summary, the prevalence of the K65R mutation was low, but its incidence has increased in recent years. The K65R mutation was associated with non-PI regimens and the didanosine–tenofovir combination, and the presence of TAM appears to protect against the K65R mutation. This mutation does not compromise the possibility of rescue.

Absence of New Thymidine-Associated Mutations and Evidence of an Immune Virologic Response Over a 12-Month Period in a Cohort of Antiretroviral-Experienced HIV-1-Infected Subjects Treated With Tenofovir Combination Therapy

Absence of New Thymidine-Associated Mutations and Evidence of an Immune Virologic Response Over a 12-Month Period in a Cohort of Antiretroviral-Experienced HIV-1-Infected Subjects Treated With Tenofovir Combination Therapy

Mechanism of Anti-Human Immunodeficiency Virus Activity of β- d -6-Cyclopropylamino-2′,3′-Didehydro-2′,3′-Dideoxyguanosine

To better understand the importance of the oxygen in the ribose ring of planar unsaturated nucleoside analogs that target human immunodeficiency virus (HIV), a 6-cyclopropyl-substituted prodrug of 2',3'-didehydro-2',3'-dideoxyguanosine (cyclo-d4G) was synthesized, and its cellular metabolism, antiviral activity, and pharmacokinetic behavior were studied. Cyclo-d4G had selective anti-HIV activity in primary blood mononuclear cells (PBMCs), effectively inhibiting the LAI strain of HIV-1 by 50% at 1.1 +/- 0.1 microM while showing 50% inhibition of cell viability at 84.5 microM. The antiviral activity in PBMCs was not markedly affected by mutations of methionine to valine at position 184 or by thymidine-associated mutations in the viral reverse transcriptase. Mutations of leucine 74 to valine and of lysine 65 to arginine had mild to moderate resistance (as high as fivefold). Studies to delineate the mechanism of cellular metabolism and activation of cyclo-d4G showed reduced potency in inhibiting viral replication in the presence of the adenosine/adenylate deaminase inhibitor 2'-deoxycoformycin, implying that the antiviral activity is due to its metabolism to the 2'-dGTP analog d4GTP. Intracellular formation of sugar catabolites illustrates the chemical and potentially enzymatic instability of the glycosidic linkage in d4G. Further studies suggest that cyclo-d4G has a novel intracellular phosphorylation pathway. Cyclo-d4G had a lower potential to cause mitochondrial toxicity than 2',3'-dideoxycytidine and 2',3'-didehydro-3'-deoxythymidine in neuronal cells. Also, cyclo-d4G had advantageous synergism with many currently used anti-HIV drugs. Poor oral bioavailability observed in rhesus monkeys may be due to the labile glycosidic bond, and special formulation may be necessary for oral delivery.

Phenotypic impact of HIV reverse transcriptase M184I/V mutations in combination with single thymidine analog mutations on nucleoside reverse transcriptase inhibitor resistance.

To analyse the impact of the M184I/V mutation and individual thymidine-associated mutations (TAM) on nucleoside reverse transcriptase inhibitor (NRTI) phenotypic susceptibility and compare these results with those obtained using commercial and public algorithms. An HIV genotypic/phenotypic database with over 27 000 samples was used to obtain the median fold change (5-95th percentile) in NRTI phenotypic susceptibility for viruses from patients containing individual TAM with or without the M184I or V mutation and for wild-type patient viruses. The resulting data indicated that in vitro, individual TAM do not have an equivalent impact on NRTI resistance, with some individual TAM having little or no impact on NRTI resistance (e.g. M41L or K219Q/E/H/R). In the presence of the M184I/V mutation, re-sensitization to some drugs, including zidovudine, stavudine and tenofovir was observed despite the presence of a TAM. For didanosine and abacavir, the presence of the M184V mutation and a single TAM did not result in a fold-change increase associated with decreased drug susceptibility. Analysis of public and commercial algorithms revealed a lack of concordance regarding the impact of these mutations, and with the observed phenotypic data. These analyses should assist in the creation of rules for genotypic drug resistance algorithms for a better reflection of the impact of individual TAM and also the impact of M184I/V on resistance. These data provide additional evidence that retaining lamivudine in those treatment regimens in which TAM can be selected may provide some therapeutic benefit by maintaining the M184V mutation.