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Development of a genotype resistance test of GP41 region for patients receiving fusion inhibitors
Presented by Lisa Morris, Australia.
Morris L.1, Greengrass V.1, Plate M.1, Crowe S.1
1Burnet Institute, Clinical Research Facility, Melbourne, Australia
Objectives: While there are commercially available laboratory tests to assess viral resistance in the protease and RT regions of the genome, there are no commercially available assays to assess resistance to the fusion inhibitor drugs. A single amino acid substitution in codons 36 to 45 in the gp41 region can reduce the efficacy of T-20. Genotypic testing of the gp41 region is needed to assess fusion inhibitor susceptibility and optimize antiretroviral regimens. Our objective was to develop and validate a genotypic resistance assay that assessed the gp41 region.
Methods: Viral RNA extraction was performed using the Viroseq genotyping assay (Abbott Diagnostics). Primers were developed from conserved sequence to amplify a 700bp segment that includes most of gp41. RT-PCR was carried out using the Roche Titan One Tube RT-PCR system. Sequencing was performed using Big Dye terminator chemistry encompassing codons 1-194 of the gp41 region. Sequences were compared to consensus sequence (NL4.3) and identified mutations interpreted using the CREST algorithm to determine antiretroviral resistance.
Results: Samples from a dilution panel of cultured HIV virus spiked into HIV-seronegative plasma at dilutions ranging from 200,000 - 200 copies/ml were amplified successfully. Patient samples of subtypes CRF01_AE, B, C, D and CRF02_AG were also able to be amplified. Sequence analysis indicated concordance of sequencing results in all samples amplified. While current literature indicates major resistance mutations for fusion inhibitor drugs occur between codons 36 to 45 of gp41, this assay amplifies most of the gp41 region allowing for extended sequencing should novel mutations be identified in other regions of gp41.
Conclusions: Preliminary investigations indicate we have developed a molecular based assay for sequencing the gp41 region of the genome which is sensitive to below 2000 copies/ml HIV RNA (similar to the Viroseq assay). Roche Products Pty Limited funded development of this assay.
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