title: Cross-clade simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase inhibitor mutations by Illumina MiSeq.
abstract: BACKGROUND
  Viral resistance to antiretroviral therapy threatens our best methods to control and prevent HIV infection. Current drug resistance genotyping methods are costly, optimized for subtype B virus, and primarily detect resistance mutations to protease and reverse transcriptase inhibitors. With the increasing use of integrase inhibitors in first-line therapies, monitoring for integrase inhibitor drug resistance mutations is a priority. We designed a universal primer pair to PCR amplify all major group M HIV-1 viruses for genotyping using Illumina MiSeq to simultaneously detect drug resistance mutations associated with protease, nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, and integrase inhibitors.
  
  
  RESULTS
  A universal primer pair targeting the HIV pol gene was used to successfully PCR amplify HIV isolates representing subtypes A, B, C, D, CRF01_AE and CRF02_AG. The universal primers were then tested on 62 samples from a US cohort of injection drug users failing treatment after release from prison. 94% of the samples were successfully genotyped for known drug resistance mutations in the protease, reverse transcriptase and integrase gene products. Control experiments demonstrate that mutations present at ≥ 2% frequency are reliably detected and above the threshold of error for this method. New drug resistance mutations not found in the baseline sample were identified in 54% of the patient samples after treatment failure. 86% of patients with major drug resistance mutations had 1 or more mutations associated with drug resistance to the treatment regimen at the time point of treatment failure. 59% of the emerging mutations were found at frequencies between 2% and 20% of the total sequences generated, below the estimated limit of detection of current FDA-approved genotyping techniques. Primary plasma samples with viral loads as low as 799 copies/ml were successfully genotyped using this method.
  
  
  CONCLUSIONS
  Here we present an Illumina MiSeq-based HIV drug resistance genotyping assay. Our data suggests that this universal assay works across all major group M HIV-1 subtypes and identifies all drug resistance mutations in the pol gene known to confer resistance to protease, reverse transcriptase and integrase inhibitors. This high-throughput and sensitive assay could significantly improve access to drug resistance genotyping worldwide.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. dmdudley@wisc.edu.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Dudley
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. albailey@wisc.edu.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Bailey
authors.affiliation: Department of Epidemiology, Epidemiology and Oncology, Johns Hopkins University, Baltimore, MD, USA. shmehta@jhsph.edu.
authors.forename: Shruti H
authors.initials: SH
authors.lastname: Mehta
authors.affiliation: Department of Biology, University of South Carolina, Columbia, South Carolina, USA. austin@biol.sc.edu.
authors.forename: Austin L
authors.initials: AL
authors.lastname: Hughes
authors.affiliation: Departments of Medicine, Epidemiology and Oncology, Johns Hopkins University, Baltimore, MD, USA. gkirk@jhsph.edu.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Kirk
authors.affiliation: Department of Medicine, Division of Infectious Disease, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. rpw@medicine.wisc.edu.
authors.forename: Ryan P
authors.initials: RP
authors.lastname: Westergaard
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. dhoconno@wisc.edu.
authors.forename: David H
authors.initials: DH
authors.lastname: O'Connor
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology / Q000627:therapeutic use; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D060005:Genotyping Techniques / Q000379:methods*; D015658:HIV Infections / Q000821:virology; D019427:HIV Integrase / Q000235:genetics; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D019380:Anti-HIV Agents; D017931:DNA Primers; D019427:HIV Integrase; D054303:HIV Reverse Transcriptase; D016333:HIV Protease