title: Decreased phenotypic susceptibility to etravirine in patients with predicted genotypic sensitivity.
abstract: BACKGROUND
  A sensitive, phenotypic reverse transcriptase (RT)-based drug susceptibility assay for the detection of etravirine (ETR) resistance in patient isolates was developed and compared with the results from direct sequencing and ultra-deep pyrosequencing (UDPS).
  
  
  METHODS
  Samples were obtained from 15 patients with antiretroviral therapy (ART) failure and from five non-nucleoside reverse transcriptase inhibitor (NNRTI)-naïve patients of whom four were infected by an NNRTI-resistant strain (transmitted drug resistance, TDR). In five patients, two consecutive samples (a and b) were taken for follow up of the virological response. HIV-1 RT was purified and drug susceptibility (IC50) to ETR was estimated. Direct sequencing was performed in all samples and UDPS in samples from nine patients.
  
  
  RESULTS
  Increased IC50 to ETR was found in samples from 13 patients where direct sequencing predicted resistance in only four. UDPS identified additional (N = 11) NNRTI resistance associated mutations (RAMs) in six of nine tested patients. During early failure, IC50 increases were observed in three of six patients without any ETR-RAMs detected by direct sequencing. In further two patients, who stopped NNRTI before sampling, increased IC50 values were found shortly after, despite absence of ETR-RAMs. In two patients who had stopped NNRTI for >1 year, a concordance between phenotype and genotypes was found. Two patients with TDR had increased IC50 despite no ETR-RAMs were detected by direct sequencing. UDPS revealed additional ETR-RAMs in four patients with a discrepancy between phenotype and direct sequencing.
  
  
  CONCLUSIONS
  The RT-based phenotypic assay showed decreased ETR susceptibility in patients where direct sequencing predicted ETR-sensitive virus. This increased phenotypic sensitivity was to a large extent supported by UDPS and treatment history. Our method could be valuable for further studies on the phenotypic kinetics of NNRTI resistance. The clinical relevance remains to be studied in larger patient-populations.
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Agneskog
authors.affiliation: Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Piotr
authors.initials: P
authors.lastname: Nowak
authors.affiliation: Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Catharina
authors.initials: C
authors.lastname: Maijgren Steffensson
authors.affiliation: Institut de Recerca de la SIDA irsiCaixa, Hospital Universitari Germans Trias I Pujol, Universitat Autònoma de Barcelona, Catalonia, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Casadellà
authors.affiliation: Institut de Recerca de la SIDA irsiCaixa, Hospital Universitari Germans Trias I Pujol, Universitat Autònoma de Barcelona, Catalonia, Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Noguera-Julian
authors.affiliation: Institut de Recerca de la SIDA irsiCaixa, Hospital Universitari Germans Trias I Pujol, Universitat Autònoma de Barcelona, Catalonia, Spain.
authors.forename: Roger
authors.initials: R
authors.lastname: Paredes
authors.affiliation: Cavidi AB, Uppsala, Sweden.
authors.forename: Clas F R
authors.initials: CF
authors.lastname: Källander
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Sönnerborg
mesh_terms: D000328:Adult; D019380:Anti-HIV Agents / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D005838:Genotype; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D008875:Middle Aged; D009570:Nitriles; D010641:Phenotype; D011724:Pyridazines / Q000494:pharmacology*; D011743:Pyrimidines; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D009570:Nitriles; D011724:Pyridazines; D011743:Pyrimidines; D018894:Reverse Transcriptase Inhibitors; C451734:etravirine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase