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17th International HIV Drug Resistance Workshop10-14 June 2008, Sitges, Spain |
IMPROVED DETECTION OF X4 VIRUS BY V3 GENOTYPING: APPLICATION TO PLASMA RNA AND PROVIRAL DNA
Antivir Ther. 2008; 13(Suppl. 3):A99 (abstract no. 89)
A Moores1, A Thielen2, W Dong1, A Low1, C Woods1, M Jensen3, B Wynhoven1, D Chan1, C Glascock1 and PR Harrigan1
1BC Centre for Excellence in HIV/AIDS, Vancouver Canada; 2Max-Planck Institute for Informatics, Saarbrucken, Germany; 3University of Georgia, Athens, GA, USA
BACKGROUND: It would be advantageous to be able to screen patients for HIV tropism by genotypic methods, but previous analyses showed that standard genotype methods detect as few as 30–50% of X4 or dual/mixed virus in clinical samples. In addition, it would be useful to be able to test for tropism when plasma viral load (pVL) is undetectable.
METHODS: Triplicate independent V3 genotype determinations were performed using custom alignment and base-calling software (RE-call) with no manual intervention in 63 samples, in which tropism phenotype was previously determined using the Monogram Trofile assay. HIV tropism was predicted from V3 genotype using protein-specific scoring matrix (PSSM) and/or geno2pheno. In addition, proviral HIV DNA V3 sequence was assessed in 26 R5/X4 and 14 R5 individuals after pVL became undetectable as a result of standard highly active antiretroviral therapy.
RESULTS: This approach led to increased sensitivity and specificity compared with previous population-based V3 approaches. Using a combination of the PSSM and geno2pheno methods, sensitivity and specificity increased to 75.8% and 91.1%, respectively. Furthermore, the Monogram X4 luciferase readout in the phenotypic assay was correlated to the ‘scores’ provided by each genotypic predictor (for example, PSSM, R2=0.54 P<0.001), suggesting that V3 sequence variation alone explains much of the variation in the X4 phenotype parameter. Of samples with undetectable pVL, the sensitivity and specificity using proviral DNA V3 sequence for predicting pre-therapy Trofile results was 76% and 71% for PSSM or 77% and 93% for geno2pheno, respectively. Further assessment of these methodologies using a blinded analysis of an independent HIV RNA dataset (n>300) and also using 454 sequencing is underway.
CONCLUSIONS: Fully automated analyses of multiple V3 sequence assessments detect a much greater proportion of X4 samples than previously possible, regardless of the genotype algorithm used. Using this approach, most patients with X4 virus can be screened out, at a cost of approximately $300–400 per assay. The ability to measure tropism from proviral DNA suggests the possibility of screening for those with suppressed pVL who may wish to switch to CCR5 antagonists for tolerability or other reasons.
2008-06-10
89
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