3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

RAPID AND SIMPLE METHOD TO DETERMINE DRUG SUSCEPTIBILITY OF CLINICAL HIV-1 ISOLATES

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:25 (abstract no. 24)

Nicole K.T. Back, Toon van Bommel, Dorien de Jong, Pauline Schipper, Menno de Jong and Charles A.B. Boucher
Antiviral Therapy Laboratory, Department of Virology, University of Amsterdam, Amsterdam, The Netherlands

AIM: To develop a rapid and simple method for evaluation of drug susceptibility of clinical samples.

METHODS: Recombinant HXB-2 viruses chimeric for the RT gene were generated by using the recently described recombinant virus assay (RVA) (Kellam et al., Antimicrob Agents Chemother. 1994 Jan;38(1):23-30).. Briefly, PCR amplified RT fragments from patient PBMCs were electroporated in a T-cell line together with a molecular HXB-2 clone lacking the RT fragment. This will result in recombination between the overlapping sequences of the RT fragment and the deletion clone. The generated recombinant syncytium-inducing viruses were subsequently tested for their drug susceptibility in the MTT assay (Pauwels et al., J Virol Methods. 1988 Aug;20(4):309-21). This method is a colorimetric cell-killing assay that monitors the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide (MTT) to a blue formazan product, which can be easily detected by the spectrophotometer.

RESULTS: To validate the RVA/MTT assay we used two recombinant viruses containing either the RT fragment of HXB2 (wild type) or this fragment carrying zidovidine resistance mutations at amino acid positions 67,70,215,219 (RTMC). The zidovidine sensitivity for both recombinant viruses was assessed. The ratio of the 50% inhibitory concentrations (IC₅₀) for the wild type and mutant virus was similar using the RVA/MTT assay to the ratio observed with the RVA/HeLa CD4+ plaque assay using a virus inoculum of 0.001 m.o.i. Formazan production was determined after 5 days culture on MT2 cells. Repeated measurement demonstrated reproducible results (1.17 log variation in IC₅₀ for wild type virus). In addition, both sensitivity assays were compared using large numbers of nevirapine treated clinical HIV-1 isolates. Both gave reproducible trends in IC₅₀'s.

CONCLUSIONS: The combination of the recombinant virus assay using a syncytium inducing deletion clone (HXB2RT) with the MTT assay provides a rapid and simple format with automated read-out for drug susceptibility testing of large numbers of clinical samples.

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1994-08-02
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