Phenotypic and genotypic analyses to guide selection of reverse transcriptase inhibitors in second-line HIV therapy following extended virological failure in Uganda.

OBJECTIVES: We investigated phenotypic and genotypic resistance after 2 years of first-line therapy with two HIV treatment regimens in the absence of virological monitoring. METHODS: NORA [Nevirapine OR Abacavir study, a sub-study of the Development of AntiRetroviral Therapy in Africa (DART) trial] randomized 600 symptomatic HIV-infected Ugandan adults (CD4 cell count <200 cells/mm(3)) to receive zidovudine/lamivudine plus abacavir (cABC arm) or nevirapine (cNVP arm). All virological tests were performed retrospectively, including resistance tests on week 96 plasma samples with HIV RNA levels ≥1000 copies/mL. Phenotypic resistance was expressed as fold-change in IC(50) (FC) relative to wild-type virus. RESULTS: HIV-1 RNA viral load ≥1000 copies/mL at week 96 was seen in 58/204 (28.4%) cABC participants and 21/159 (13.2%) cNVP participants. Resistance results were available in 35 cABC and 17 cNVP participants; 31 (89%) cABC and 16 (94%) cNVP isolates had a week 96 FC below the biological cut-off for tenofovir (2.2). In the cNVP arm, 16/17 participants had resistance mutations synonymous with high-level resistance to nevirapine and efavirenz; FC values for etravirine were above the biological cut-off in 9 (53%) isolates. In multivariate regression models, K65R, Y115F and the presence of thymidine analogue-associated mutations were associated with increased susceptibility to etravirine in the cABC arm. CONCLUSIONS: Our data support the use of tenofovir following failure of a first-line zidovudine-containing regimen and shed further light on non-nucleoside reverse transcriptase inhibitor hypersusceptibility.

The impact of individual human immunodeficiency virus type 1 protease mutations on drug susceptibility is highly influenced by complex interactions with the background protease sequence.

The requirement for multiple mutations for protease inhibitor (PI) resistance necessitates a better understanding of the molecular basis of resistance development. The novel bioinformatics resistance determination approach presented here elaborates on genetic profiles observed in clinical human immunodeficiency virus type 1 (HIV-1) isolates. Synthetic protease sequences were cloned in a wild-type HIV-1 background to generate a large number of close variants, covering 69 mutation clusters between multi-PI-resistant viruses and their corresponding genetically closely related, but PI-susceptible, counterparts. The vast number of mutants generated facilitates a profound and broad analysis of the influence of the background on the effect of individual PI resistance-associated mutations (PI-RAMs) on PI susceptibility. Within a set of viruses, all PI-RAMs that differed between susceptible and resistant viruses were varied while maintaining the background sequence from the resistant virus. The PI darunavir was used to evaluate PI susceptibility. Single sets allowed delineation of the impact of individual mutations on PI susceptibility, as well as the influence of PI-RAMs on one another. Comparing across sets, it could be inferred how the background influenced the interaction between two mutations, in some cases even changing antagonistic relationships into synergistic ones or vice versa. The approach elaborates on patient data and demonstrates how the specific mutational background greatly influences the impact of individual mutations on PI susceptibility in clinical patterns.

Nine-year trends in clinically relevant reduced susceptibility of HIV-1 to antiretrovirals.

BACKGROUND: Temporal changes in HIV-1 resistance both reflect and influence the clinical use of antiretrovirals (ARVs). OBJECTIVE: To determine temporal trends in reduced susceptibility to ARVs and resistance mutations in routine clinical samples (RCS) from HIV infected patients. STUDY DESIGN: Calculated fold-changes (FC) for ARVs were determined for viral genotypes from RCS received between July 1998 and June 2007 using vircoTYPE HIV-1 (Version 4.2.01). The prevalence of isolates with clinically relevant reduced susceptibility and of resistance mutations were determined for consecutive 6-month periods. RESULTS: 242,003 RCS were identified. The prevalence of RCS exhibiting reduced susceptibility to > or =1 drug among any of three ARV classes decreased from 82% to 66.5% and of these, to > or =1 drug in each of the three ARV classes and from 30.5% to 15.7% from December 98 to June 07 (p< or =0.0001). The prevalence of mutations associated with NRTI, NNRTI and PI resistance generally reflected evolving use of these drug classes. Among fully susceptible RCS, isolates that exhibited resistance mutations were rare. CONCLUSIONS: Clinically relevant reduced susceptibility to ARVs declined over the 9-year period. There was a general higher prevalence of reduced susceptibility to RT inhibitors than to PIs.

The HIV-1 protease resistance mutation I50L is associated with resistance to atazanavir and susceptibility to other protease inhibitors in multiple mutational contexts.

BACKGROUND: The HIV-1 protease mutation I50 L causes atazanavir resistance but increases susceptibility to other PIs. Predicted phenotypic FC values were obtained from viral genotypes, using the virtual Phenotype-LM bioinformatics tool (powering vircoTYPE). OBJECTIVE: To evaluate I50 L's effect on susceptibility to 8 PIs, in a large genotype database. STUDY DESIGN: I50 L containing routine clinical isolate samples in Virco's genotype database were paired with samples having like patterns (or profiles) of IAS-USA-defined primary PI mutations, but lacking I50 L. Using vircoTYPE (version 4.1), the median predicted FC for each mutational profile was determined. I50 L-associated shifts in FC were evaluated using drug-specific CCOs. RESULTS: We selected 307 and 37098 samples with and without I50 L. These corresponded to 31 mutation patterns of > or =3 samples each. I50 L caused resistance to atazanavir in all 31 mutation contexts, but was associated with higher susceptibility for other PIs. The largest I50 L-associated shifts in median predicted FC were: 1.2 to 42.4 (atazanavir), 10.2 to 3.2 (amprenavir), 3.3 to 0.5 (darunavir), 13 to 0.5 (indinavir), 34.9 to 1.3 (lopinavir), 22.3 to 1.3 (nelfinavir), 5.2 to 0.3 (saquinavir) and 29.9 to 5.2 (tipranavir). CONCLUSIONS: The PI mutation I50 L causes clinically relevant resistance and increased susceptibility to atazanavir and other PIs respectively.

Novel drug resistance pattern associated with the mutations K70G and M184V in human immunodeficiency virus type 1 reverse transcriptase.

We describe an unusual pathway of human immunodeficiency virus type 1 reverse transcriptase resistance during therapy with tenofovir-emtricitabine, characterized initially by the mutations K70E and M184V and later by K70G and M184V, with the two mutations coexisting on the same viral genome. Phenotypic resistance to lamivudine, emtricitabine, abacavir, didanosine, and tenofovir was observed, whereas susceptibility to zidovudine and stavudine was preserved.

Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400.

A Pseudomonas fluorescens Tn5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis. Since this mutant was leaky, a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor delta-aminolevulinate (ALA). Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth. A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin. The DeltahemH mutant was characterized and the corresponding hemH gene sequenced. Antibodies against P. fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant. The DeltahemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans. These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production.

Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter.

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron- and pH-inducible lipoprotein gene sfbA, which is a component of a novel ABC-type transporter system required for virulence. This gene is located on a 4 kb Salmonella-specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild-type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.