249   Validation of the Ultrasensitive HIV-1 Genotyping Assay.

S. Y. Lee*, M. Sandhu, M. P. Griswold, and M. Van Gorder.
Consolidated Lab. Services, Van Nuys, CA.

Background:The sensitivities of the quantitative viral assays are now down to 50 cps/mL. Most commercially available genotyping assays require a plasma concentration greater than 1,000 cps/mL. Therefore, there is a need to have reliable genotyping tests for viral load between 50—1,000 cps/mL, which may portend treatment failure. Visible Genetics Inc (VGI) TRUGENE HIV-1 genotyping kit using CLIP chemistry, a bi-directional cycle sequencing technology, can allow users to perform genotyping assays with low viral load. To assess the availability for ultrasensitive application with patient samples, we verified the assay with low viral load on sensitivity, linearity, and reproducibility.

Methods:Viral RNAs were extracted using spin columns (Qiagen) with modification by centrifuging 1 mL of plasma at 25,500 × g for 1 hour at 4(C. Plasma HIV RNA levels were measured using the Roche Amplicor HIV- 1 Monitor (standard/ultrasensitive) kit. The OpenGene system and TRUGENE HIV-1 kit were used for genotyping. The assay was performed on a total of 84 specimens obtained from patients on HAART with viral load less than 1,000 cps/mL. One sample (HIV-1 RNA 2.0 × 10⁶cps/mL) was serially diluted with normal plasma to generate controls to monitor linearity. Genotyping of five samples was performed on three separate runs to confirm reproducibility.

Results:Of 84 specimens, 77 samples were successfully genotyped; 7 samples with viral load less than 100 cps/ mL failed. The lowest viral load we effectively genotyped was 116 cps/mL. The sequencing signals retained intensities similar to that achieved with high viral load (>1,000 cps/mL). This further validates the reliability of the procedure. 62 samples (80%) contained viruses that expressed major resistance codons in the protease and/or reverse transcriptase genes. The assay showed excellent linearity from 2.0 × 10⁶down to 10²cps/mL. 100% reproducibility was observed among those five samples.

Conclusions:Our data demonstrate that TRUGENE HIV-1 kit could successfully perform genotyping for patient samples with viral load between 116—1,000 cps/mL. High prevalence of resistance codons identified in

this group supports the merits for potential clinical use. The long-term benefits of this ultrasensitive procedure on patients are currently under investigation.