title: Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis.
abstract: BACKGROUND
  Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage.
  
  
  METHODS
  Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor].
  
  
  RESULTS
  Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate.
  
  
  CONCLUSIONS
  The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.
authors.affiliation: Institute of Immunology and Genetics, Pfaffplatz 10, 67655 Kaiserslautern, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Däumer
authors.forename: Rolf
authors.initials: R
authors.lastname: Kaiser
authors.forename: Rolf
authors.initials: R
authors.lastname: Klein
authors.forename: Thomas
authors.initials: T
authors.lastname: Lengauer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Thiele
authors.forename: Alexander
authors.initials: A
authors.lastname: Thielen
mesh_terms: D005838:Genotype; D015658:HIV Infections / Q000235:genetics / Q000821:virology; D015497:HIV-1 / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D010641:Phenotype; D019718:Receptors, CXCR4 / Q000235:genetics; D056189:Viral Tropism / Q000235:genetics*
chemical_list: D019718:Receptors, CXCR4