title: Evaluation of a benchtop HIV ultradeep pyrosequencing drug resistance assay in the clinical laboratory.
abstract: Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had low-abundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.
authors.affiliation: Laboratory for Viruses and Molecular Biology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel. bavidor@tasmc.health.gov.il
authors.forename: Boaz
authors.initials: B
authors.lastname: Avidor
authors.forename: Shirley
authors.initials: S
authors.lastname: Girshengorn
authors.forename: Natalia
authors.initials: N
authors.lastname: Matus
authors.forename: Hadass
authors.initials: H
authors.lastname: Talio
authors.forename: Svetlana
authors.initials: S
authors.lastname: Achsanov
authors.forename: Irene
authors.initials: I
authors.lastname: Zeldis
authors.forename: Ilana S
authors.initials: IS
authors.lastname: Fratty
authors.forename: Eugene
authors.initials: E
authors.lastname: Katchman
authors.forename: Tal
authors.initials: T
authors.lastname: Brosh-Nissimov
authors.forename: David
authors.initials: D
authors.lastname: Hassin
authors.forename: Danny
authors.initials: D
authors.lastname: Alon
authors.forename: Zvi
authors.initials: Z
authors.lastname: Bentwich
authors.forename: Israel
authors.initials: I
authors.lastname: Yust
authors.forename: Sharon
authors.initials: S
authors.lastname: Amit
authors.forename: Relly
authors.initials: R
authors.lastname: Forer
authors.forename: Ina
authors.initials: I
authors.lastname: Vulih Shultsman
authors.forename: Dan
authors.initials: D
authors.lastname: Turner
mesh_terms: D000328:Adult; D000368:Aged; D019295:Computational Biology / Q000379:methods*; D024882:Drug Resistance, Viral; D005260:Female; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008875:Middle Aged; D050505:Mutant Proteins / Q000235:genetics; D020125:Mutation, Missense; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods; D055815:Young Adult
chemical_list: D050505:Mutant Proteins; D014764:Viral Proteins