title: Deep Sequencing of HIV-1 RNA and DNA in Newly Diagnosed Patients with Baseline Drug Resistance Showed No Indications for Hidden Resistance and Is Biased by Strong Interference of Hypermutation.
abstract: Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant.
authors.affiliation: AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Kenny
authors.initials: K
authors.lastname: Dauwe
authors.affiliation: AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Delfien
authors.initials: D
authors.lastname: Staelens
authors.affiliation: AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Vancoillie
authors.affiliation: AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Virginie
authors.initials: V
authors.lastname: Mortier
authors.affiliation: AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium chris.verhofstede@ugent.be.
authors.forename: Chris
authors.initials: C
authors.lastname: Verhofstede
mesh_terms: D004279:DNA, Viral / Q000235:genetics*; D024882:Drug Resistance, Viral; D005260:Female; D015658:HIV Infections / Q000821:virology*; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics*; D012189:Retrospective Studies
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1