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Education > Clinical Updates

Clinical Update #4 - News from the 16^th International HIV Drug Resistance Workshop

Barbados, West Indies
June 12-16, 2007

Assessment of the Performance of the VircoTYPE System in an Independent Clinical Dataset B.Winters, J. Montaner, P.R. Harrigan, B. Gazzard, A. Pozniak, K. Van Der Borght, J. Villacian, and L. Bacheler. Poster 155

An important aspect of the development of any system that predicts response to antiretroviral therapy is its validation in a diverse clinical dataset not used for algorithm development. Bart Winters and colleagues from Virco, in collaboration with the British Columbia Centre for Excellence in HIV/AIDS and the Chelsea and Westminster Healthcare NHS Trust, presented a poster at the 16^th International HIV Drug Resistance Workshop evaluating the accuracy of the vircoTYPE (vT) system in predicting virologic response to antiretroviral therapy in an unseen data set compared with 3 genotypic interpretation algorithms.

This retrospective analysis utilized a dataset composed of previously collected validation data that had never been used for algorithm development combined with recent updates from the British Columbia and Chelsea and Westminster cohorts (n=2108 Treatment Change Episodes). The 4 resistance interpretation systems evaluated were: HIVdb (Stanford version 4.2.6), ANRS (August 2006), REGA (version 6.4.1), and vircoTYPE (version 4.0.01).

Baseline sensitivity scores for each treatment regimen by each interpretation system were calculated as the number of active drugs in the regimen. Response was defined as a >1 log₁₀ drop in HIV RNA levels from baseline or an HIV RNA level below the limit of detection at week 8 and/or 24. The association of baseline sensitivity scores with virologic outcome was evaluated using 3 metrics: namely, Pearson Correlation, diagnostic accuracy (as area under the ROC curve [AUC]), and Responder Non-responder Classification. Separate analyses were performed for the entire unseen dataset and for subgroups of patients with a difference of >0.5 in sensitivity score between vT and each of the algorithms.

Of the resistance algorithms evaluated, the vT based sensitivity score was most strongly associated with virologic response at week 8 in the entire unseen data set. When focusing on those regimens for which resistance interpretations varied, vT was again more correlated with response. At week 24, results differed based on the handling of missing data: if drop-outs were excluded from analysis, the vT performance observed at week 8 persisted up to week 24; if all drop-outs for any reason were considered virologic non-responders, the performance of all algorithms decreased dramatically and no relevant differences were observed.

The authors concluded that these data confirm, in an unseen clinical outcome dataset, the higher accuracy of the vircoTYPE system in predicting 8-week outcome. Additionally, the vT system compared favorably with other algorithms at predicting week 24 outcomes as well. If you are interested in viewing the complete poster, please click here

Detection limit of X4 minority virus in abundant R5-tropic virus populations in standard genotypic and phenotypic assays I Vandenbroucke, V Van Eygen, E Rondelez, K Van Baelen, and LJ Stuyver. Poster 139

The CCR5 antagonists represent a new class of antiretroviral therapy for patients with multidrug-resistant HIV. The clinical response to this new class of agents is partially driven by absence of X4 viruses; however, no clear cut-off values on presence/absence of such virus population have been established. Among the first steps in establishing such cut-off values is determining the detection limit of X4 viruses in an abundant R5 virus population at various HIV RNA levels.

Ina Vandenbroucke and colleagues from Virco presented a poster at the 16th International HIV Drug Resistance Workshop reporting on the sensitivity of a clonal genotypic and population phenotypic tropism assay developed by Virco. Artificial mixtures of R5-tropic (rec JRCSF) and X4-tropic (rec NL4.3) recombinant virus stocks were prepared to create various minority X4 inputs at differing HIV RNA concentrations as follows:

• 2.5%/97.5% and 1%/99% X4/R5 at HIV RNA level of 5 log₁₀ IU/mL
• 20%/80% and 8%/92% X4/R5 at HIV RNA level of 4 log₁₀ IU/mL
• 20%/80% X4/R5 at HIV RNA level of 3 log₁₀ IU/mL

Theoretical analysis illustrated that the ability to detect minority quasispecies depended on initial viral load levels and assay design. Experimentally,

Using a clonal genotypic approach:

• at 5 log IU/ml- a 1% X4 fraction was detected in 1/3 experiments when sequencing ~95 clones; a 2.5% minority variant was detected in 4/6 repeated experiments
• at 4 log IU/ml- the detection success rate was 100% for an 8% and 20% minority population
• at 3 log IU/ml- the 20% minority variant was detected in 3/5 experiments; however the measured percentage of the minority X4 virus deviated substantially from the expected value

Using a population phenotypic analysis:

• at 5 log IU/ml- a 1% X4 minority species was detected by microscopic and Florescence Activated Cell Sorting (FACS) analysis; in the 2.5% X4 experiment in which no X4 could be detected genotypically, there was also no X4-tropic virus detected by phenotypic analysis
• at 4 log IU/ml- X4 minority species were detected in all experiments
• at 3 log IU/ml- X4 minority species were detected in 2/3 experiments

The authors concluded that the population phenotypic assay provided a fast alternative for clonal genotypic analysis, although the latter is more sensitive and generates the most information. As theoretically anticipated, the ability to detect minority X4 virus strains decreased at lower HIV RNA levels and minority species input. If you are interested in viewing the complete poster, please click here.

Low Level of Baseline Resistance to Integrase Inhibitors L731,988 and L870,810 in Randomly Selected SubType B and Non-B HIV-1 Strains K Van Baelen, M Clynhens, E Rondelez, V Van Eygen, P Van Den Zegel, H Vermeiren, I VandenBroucke and L J Stuyver. Poster 5
HIV-1 integrase inhibitors (INIs) represent a promising new class of antiretroviral therapy currently undergoing phase II/III clinical trials. These agents block the integration of proviral DNA into the cellular genome. Virco has developed an experimental phenotypic assay which incorporates patient sample-derived Reverse Transcriptase (RT), RNaseH, and Integrase (IN) genes. Kurt Van Baelen and colleagues from Virco presented a poster at the 16^th International HIV Drug Resistance Workshop utilizing this assay to determine the effects of naturally occurring polymorphisms in the IN gene on susceptibility to two experimental INIs (L731,988 and L870,810) as well as to darunavir (DRV), as a control. In this analysis, 89 clonal recombinant virus stocks derived from 47 samples from integrase inhibitor-naïve patients were selected for genotypic and phenotypic analysis. Wild type HXB2D and amplicons containing the T66I or E92Q integrase inhibitor resistance associated mutations (INI RAMs) were used as controls. The results were as follows:

• The IC50 for HXB2D were 1.5+0.3µM for L731,988, 2.8+0.6nM for L870,810, 3.9+0.9nM for DRV
• Phenotypic testing of E92Q showed a ~6 fold and ~10 fold decrease in susceptibility to L731,988 and L870,810 respectively; T66I showed ~2.5 fold decrease in susceptibility to L731,988 while no FC effect was observed in susceptibility to L870,810; neither mutant showed decreased susceptibility to DRV
• In general, patient derived recombinant stocks showed no baseline resistance to both INIs L731,988 and L870,810

The IC₅₀ values for HXB2D and FC values for E92Q and T66I integrase inhibitor mutant viruses were in concordance with earlier observations. Although several IN polymorphisms known to be associated with INI resistance were detected by genotypic analysis of patient samples (V72I, L74I, T97A, A128T, Q148H, V151I, K156N, E157Q, V165I, V201I, I203M, T206S, S230N) , these amino acid changes did not have significant effect on susceptibility to the experimental INIs tested. Comparison of IC₅₀ and FC values of recombinant clones containing identical RT-IN or IN sequences showed the overall reproducibility of the antiviral experiments.
The authors concluded that this Virco-developed assay which contains the RT-RNaseH-IN genes should allow for the study of the clinical significance of naturally occurring polymorphisms as well as NRTI, NNRTI, and INI selected variants. If you are interested in viewing the complete poster, please click here.

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