title: Longitudinal Detection and Persistence of Minority Drug-Resistant Populations and Their Effect on Salvage Therapy.
abstract: BACKGROUND
  Drug-resistant HIV are more prevalent and persist longer than previously demonstrated by bulk sequencing due to the ability to detect low-frequency variants. To clarify a clinical benefit to monitoring minority-level drug resistance populations as a guide to select active drugs for salvage therapy, we retrospectively analyzed the dynamics of low-frequency drug-resistant population in antiretroviral (ARV)-exposed drug resistant individuals.
  
  
  METHODS
  Six HIV-infected individuals treated with ARV for more than five years were analyzed. These individuals had difficulty in controlling viremia, and treatment regimens were switched multiple times guided by standard drug resistance testing using bulk sequencing. To detect minority variant populations with drug resistance, we used a highly sensitive allele-specific PCR (AS-PCR) with detection thresholds of 0.3-2%. According to ARV used in these individuals, we focused on the following seven reverse transcriptase inhibitor-resistant mutations: M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y. Results of AS-PCR were compared with bulk sequencing data for concordance and presence of additional mutations. To clarify the genetic relationship between low-frequency and high-frequency populations, AS-PCR amplicon sequences were compared with bulk sequences in phylogenetic analysis.
  
  
  RESULTS
  The use of AS-PCR enabled detection of the drug-resistant mutations, M41L, K103N, Y181C, M184V and T215Y, present as low-frequency populations in five of the six individuals. These drug resistant variants persisted for several years without ARV pressure. Phylogenetic analysis indicated that pre-existing K103N and T215I variants had close genetic relationships with high-frequency K103N and T215I observed during treatment.
  
  
  CONCLUSIONS
  Our results demonstrate the long-term persistence of drug-resistant viruses in the absence of drug pressure. The rapid virologic failures with pre-existing mutant viruses detectable by AS-PCR highlight the clinical importance of low-frequency drug-resistant viruses. Thus, our results highlight the usefulness of AS-PCR and support its expanded evaluation in ART clinical management.
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Nishizawa
authors.affiliation: Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Hattori
authors.affiliation: Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Teiichiro
authors.initials: T
authors.lastname: Shiino
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Matano
authors.affiliation: Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Walid
authors.initials: W
authors.lastname: Heneine
authors.affiliation: Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Johnson
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan; Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan; Department of AIDS Research, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Sugiura
mesh_terms: D000483:Alleles; D023241:Antiretroviral Therapy, Highly Active; D018791:CD4 Lymphocyte Count; D024882:Drug Resistance, Viral; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy* / Q000453:epidemiology / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D018451:Homosexuality, Male; D006801:Humans; D007564:Japan / Q000453:epidemiology; D008297:Male; D055118:Medication Adherence; D009154:Mutation; D059645:Mutation Rate; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016879:Salvage Therapy; D019562:Viral Load