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

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

Nov 01, 2022 298 likes •420 views

Tropism Testing with XTrack C Heinz Stucki Institut fr med. Mikrobiologie Universitt Basel Introduction Genotyping: typically at amino acid level CTRPSNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC e.g. 11/25 and G2P XTrack C : at nucleic acid

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SLIDE 1

Tropism Testing with XTrackC

Heinz Stucki Institut für med. Mikrobiologie Universität Basel

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SLIDE 2

Introduction

  • Genotyping:

typically at amino acid level

CTRPSNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC

  • XTrackC: at nucleic acid level

TGTACAAGACCCAGCAACAATACAAGAAAAAGTATACATATAGGACCAGGGAGAGCATTTTATACAACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGT

Hybridization to R5 reference sequence

Level of hybridization determines formation of different structures

  • resolved by capillary electrophoresis

Homoduplex Heteroduplex e.g. 11/25 and G2P

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SLIDE 3

Validation

  • Study of SHCS: samples for validation: 100
  • 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

  • Comparison of results with Trofile assay
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SLIDE 4

Phylogenetic tree of HIV1 DNA sequences

Source: Los Alamos database 2008

blue: R5 tropic red: X4 tropic

  • 5 main lobes
  • X4 strains more outside
  • some lobes show X4 clusters
  • closely related?
  • dual?

Probe design

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SLIDE 5

Dendrogram synthesis of four specific R5-probes Representation

BUT to date:

  • no general representation in Los Alamos DB!
  • “Sequences of interest” seem predominant

more “real life material” needed!

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SLIDE 6

Profile of patient’s virus Results for R5 or X4 tropic viruses / mixed populations

homoduplex MW ss probe

CCR5 tropic CXCR4 tropic Mixed population

heteroduplex MW ss probe ss probe heteroduplex MW homoduplex

R5-window

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SLIDE 7

Issues Example for a sample with strange migration

Heteroduplex peak located in unexpected position Co-receptor tropism not assignable

ss probe heteroduplex homoduplex MW

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SLIDE 8
  • 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

Limits of XTrackC

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SLIDE 9

Tropism Quiz

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

who is who?

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SLIDE 10

Summary XTrackC

  • 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

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SLIDE 11

Acknowledgment

Thomas Klimkait Sarah Wagner Sevèrine Louvel

Institut für medizinische Mikrobiologie

François Hamy Vincent Vidal

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SLIDE 12

Principle of rPheno

Reporter read-out

env

Inhibitor Inhibitor R5 R5

Virus Reconstitution (Plasmid) Amplification / Reinfection Patient virus Phenotype Genotype Geno2Pheno XTrackC read-out

Inhibitor Inhibitor X4 X4

amplification