Tropism Testing with XTrack C Heinz Stucki Institut fr med. - PowerPoint PPT Presentation

Tropism Testing with XTrack C Heinz Stucki Institut für med. Mikrobiologie Universität Basel

Introduction • Genotyping: typically at amino acid level CTRPSNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC e.g. 11/25 and G2P • XTrack C : at nucleic acid level TGTACAAGACCCAGCAACAATACAAGAAAAAGTATACATATAGGACCAGGGAGAGCATTTTATACAACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGT Hybridization to R5 reference sequence Homoduplex Heteroduplex Level of hybridization determines formation of different structures - resolved by capillary electrophoresis

Validation • Study of SHCS: samples for validation: 100 • Comparison of results with Trofile assay • Scheme for diagnostic Co-receptor assignment clear assignment with XTrackC 1-2 days ambiguities analyzed by genotyping (eg. 11/25, G2P) 2 days ambiguities analyzed by Phenotyping (rPheno) 3 Weeks

Probe design Phylogenetic tree of HIV1 DNA sequences • 5 main lobes •X4 strains more outside • some lobes show X4 clusters • closely related? • dual? blue: R5 tropic red: X4 tropic Source: Los Alamos database 2008

Representation Dendrogram synthesis of four specific R5-probes BUT to date: • no general representation in Los Alamos DB! • “Sequences of interest” seem predominant more “real life material” needed!

Profile of patient’s virus Results for R5 or X4 tropic viruses / mixed populations ss probe homoduplex CCR5 tropic MW ss probe heteroduplex CXCR4 tropic MW homoduplex heteroduplex Mixed population ss probe MW R5-window

Issues Example for a sample with strange migration ss probe heteroduplex homoduplex MW Heteroduplex peak located in unexpected position Co-receptor tropism not assignable

Limits of XTrack C 1. Samples of “non assignable tropism” • => sequencing • => analyze with 11/25 or Geno2Pheno Collaborators invited! 2. discordant cases; MRV resistance • expected only for low no. of samples • Phenotyping with rPheno 3. Aspects of viral fitness •rPheno in presence of drug

Tropism Quiz who is who? Patient 9 0 2 N.A. R5 R5 5 0 8 X4 X4 X4 8 1 2 R5 R5 R5 9 1 6 R5 X4 X4 6 9 6 R5 R5 X4 4 0 8 R5 X4 X4 1 6 8 R5 R5 R5 7 2 4 R5 X4 X4 4 0 4 R5 R5 N.A. 1 6 4 R5 X4 R5 1 7 2 N.A. R5 X4 1 6 1 R5 R5 X4 1 5 2 R5 X4 N.A. 7 1 5 R5 R5 X4 7 1 8 N.A. X4 X4 4 1 3 R5 R5 R5 4 0 7 R5 R5 N.A. 1 2 R5 R5 X4 1 7 X4 X4 R5 7 3 1 R5 X4 X4

Summary XTrack C • combines genotypic and structural information • fast and cost-effective • detects mixed populations • serves as starting point for genotyping • Rapid system turn-around 2d / +3d / +14d XtrackC / geno / pheno

Acknowledgment Institut für medizinische Mikrobiologie Thomas Klimkait François Hamy Vincent Vidal Sarah Wagner Sevèrine Louvel

Principle of rPheno XTrack C Geno2Pheno Genotype Inhibitor R5 R5 Inhibitor Patient virus Phenotype Reporter env read-out Virus Reconstitution (Plasmid) Amplification / Reinfection Inhibitor X4 X4 Inhibitor read-out amplification