title: Deep sequencing reveals minor protease resistance mutations in patients failing a protease inhibitor regimen.
abstract: Standard genotypic antiretroviral resistance testing, performed by bulk sequencing, does not readily detect variants that comprise <20% of the circulating HIV-1 RNA population. Nevertheless, it is valuable in selecting an antiretroviral regimen after antiretroviral failure. In patients with poor adherence, resistant variants may not reach this threshold. Therefore, deep sequencing would be potentially valuable for detecting minority resistant variants. We compared bulk sequencing and deep sequencing to detect HIV-1 drug resistance at the time of a second-line protease inhibitor (PI)-based antiretroviral regimen failure. Eligibility criteria were virologic failure (HIV-1 RNA load of >500 copies/ml) of a first-line nonnucleoside reverse transcriptase inhibitor-based regimen, with at least the M184V mutation (lamivudine resistance), and second-line failure of a lopinavir/ritonavir (LPV/r)-based regimen. An amplicon-sequencing approach on the Roche 454 system was used. Six patients with viral loads of >90,000 copies/ml and one patient with a viral load of 520 copies/ml were included. Mutations not detectable by bulk sequencing during first- and second-line failure were detected by deep sequencing during second-line failure. Low-frequency variants (>0.5% of the sequence population) harboring major protease inhibitor resistance mutations were found in 5 of 7 patients despite poor adherence to the LPV/r-based regimen. In patients with intermittent adherence to a boosted PI regimen, deep sequencing may detect minority PI-resistant variants, which likely represent early events in resistance selection. In patients with poor or intermittent adherence, there may be low evolutionary impetus for such variants to reach fixation, explaining the low prevalence of PI resistance.
authors.affiliation: Division of Medical Virology, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Randall
authors.initials: R
authors.lastname: Fisher
authors.forename: Gert U
authors.initials: GU
authors.lastname: van Zyl
authors.forename: Simon A A
authors.initials: SA
authors.lastname: Travers
authors.forename: Sergei L
authors.initials: SL
authors.lastname: Kosakovsky Pond
authors.forename: Susan
authors.initials: S
authors.lastname: Engelbrech
authors.forename: Ben
authors.initials: B
authors.lastname: Murrell
authors.forename: Konrad
authors.initials: K
authors.lastname: Scheffler
authors.forename: Davey
authors.initials: D
authors.lastname: Smith
mesh_terms: D000328:Adult; D024882:Drug Resistance, Viral; D005260:Female; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology*; D017320:HIV Protease Inhibitors / Q000008:administration & dosage* / Q000494:pharmacology; 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; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D012367:RNA, Viral; D012680:Sensitivity and Specificity; D019562:Viral Load; D055815:Young Adult
chemical_list: D017320:HIV Protease Inhibitors; D012367:RNA, Viral