1st International Workshop on HIV Drug Resistance & Treatment Strategies 25-28 June 1997, St. Petersburg, Florida, USA

PERFORMANCE CHARACTERISTICS OF PHENOTYPIC DRUG RESISTANCE TESTING (ANTIVIROGRAM™) IN MONITORING OF ANTI-HIV THERAPY

Antiviral Therapy 1997;2 (Suppl 5):28 (abstract no. 43)

K Hertogs¹, M-P de Béthune², V Miller³, B Larder⁴, S Kemp⁴ , S Bloor⁴, S Staszewski³, M Van Houtte¹, F Peeters¹ and R Pauwels^1,2
1VIRCO and ²TIBOTEC, Antwerp, Belgium, ³W Goethe Universität, Frankfurt, Germany, ⁴Glaxo Wellcome, Stevenage, UK

OBJECTIVE: Technologies to detect HIV resistance are important tools in the evaluation of new drugs and the monitoring of individualized therapy. Phenotypic resistance testing directly measures the ability of viruses to grow in the presence of a drug and is essential in establishing the relationship between a given genotype and the resulting phenotype. The performance characteristics of phenotypic resistance testing were studied.

METHODS: Recombinant virus isolates containing the combined protease (PR)-reverse transcriptase (RT) sequence (2.2 kb) of HIV-1 strains obtained from patients' plasma were used. These viruses have the advantage of being isogenic except for their RT and/or PR genes, which are of major therapeutic interest. Independently, a HeLa-CD4 plaque assay was compared with a cell viability MTT-based assay (Antivirogram™).

RESULTS: Susceptibility determinations demonstrate that a high correlation (n=43, r=0.8, P<0.00001) exists between resistance values obtained by both methods and that the results are highly reproducible within each method. However, the MTT-based method allows automation and high-throughput analysis with currently >3500 samples analysed. Furthermore, it could be demonstrated that serial dilutions of viral RNA, carrying specific resistance-associated mutations, reproducibly yielded representative phenotypic resistance data on as few as 200 copies of viral RNA. Whether RT susceptibility data are obtained from chimeric RT-only viruses or from chimeric PR-RT viruses does not significantly influence the results. Therefore it is possible to measure the resistance level to all currently used antiretroviral drugs (>10) in one standardized test. The observed phenotypic results were linked to various mutational patterns in the respective genes. These data demonstrate that one genotype can correspond with various degrees of phenotypic (cross-) resistance and that one phenotypic pattern can be the result of different complex genotypic patterns.

CONCLUSION: We have designed an in vitro system facilitating rapid and simultaneous phenotyping for all RT and PR inhibitors on a large-scale basis.

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1997-06-25
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