Background: Commercially available HIV-1 genotyping kits sequence the PR- and RT-coding regions of pol.  The TRUGENE HIV-1 gp41 ASR, with the OpenGene DNA Sequencing System, was designed to detect drug resistance mutations in the gp41-coding segment of env selected by the fusion inhibitor enfuvirtide (ENF). Assay performance was evaluated using a panel of plasma samples obtained from ENF-naive and -experienced HIV-1-infected subjects.

Methods:  Fifty-one plasma samples were obtained from 26 subjects (20 prior to ENF and 31 during or after ENF exposure).  HIV-1 RNA was extracted using a Qiagen viral RNA extraction minikit.   Samples were then subjected to an RT-PCR and the HR-1 region of env sequenced using the TRUGENE HIV-1 gp41 ASR or a “home-brew” assay using an ABI 3700 PRISM automated sequencer.  Each pair of sequences obtained was then manually aligned with each other and with a subtype B reference sequence (NL4-3), and differences between the two test sequences were scored to determine the percent agreement.  Mutations in the region including codons 36-45 were scored as possible ENF-resistance mutations. 

Results: Sequence data were obtained by both methods for 42 samples.  No sequence could be obtained for 7 samples (14%) by the home brew method and for 1 sample (2%) by the TRUGENE assay.  The mean number of ENF resistance mutations per sample was  1.0 (range, 0-3).  Mean percent agreement across a shared 231-nucleotide (nt) region encompassing HR-1 was 99.1% (range, 97.4-100%).   Mean percent agreement at the amino acid level at codons 36-45 was 97.4% (range, 80-100%).   Sequence data from multiple independent clones (n=7-14) were available for 32 samples.  Overall, there was excellent agreement between the population-based sequences obtained by the TRUGENE and home-brew methods and sequences obtained from clones. 

Conclusion: The TRUGENE HIV-1 gp41 ASRs and the OpenGene DNA Sequencing System generated highly accurate sequence data when tested with plasma samples as compared to home-brew and clonal sequence analysis.