title: Restriction fragment mass polymorphism (RFMP) analysis based on MALDI-TOF mass spectrometry for detecting antiretroviral resistance in HIV-1 infected patients.
abstract: Viral genotype assessment is important for effective clinical management of HIV-1 infected patients, especially when access and/or adherence to antiretroviral treatment is reduced. In this study, we describe development of a matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based viral genotyping assay, termed restriction fragment mass polymorphism (RFMP). This assay is suitable for sensitive, specific and high-throughput detection of multiple drug-resistant HIV-1 variants. One hundred serum samples from 60 HIV-1-infected patients previously exposed to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were analysed for the presence of drug-resistant viruses using the RFMP and direct sequencing assays. Probit analysis predicted a detection limit of 223.02 copies/mL for the RFMP assay and 1268.11 copies/mL for the direct sequencing assays using HIV-1 RNA Positive Quality Control Series. The concordance rates between the RFMP and direct sequencing assays for the examined codons were 97% (K65R), 97% (T69Ins/D), 97% (L74VI), 97% (K103N), 96% (V106AM), 97% (Q151M), 97% (Y181C), 97% (M184VI) and 94% (T215YF) in the reverse transcriptase coding region, and 100% (D30N), 100% (M46I), 100% (G48V), 100% (I50V), 100% (I54LS), 99% (V82A), 99% (I84V) and 100% (L90M) in the protease coding region. Defined mixtures were consistently and accurately identified by RFMP at 5% relative concentration of mutant to wild-type virus while at 20% or greater by direct sequencing. The RFMP assay based on mass spectrometry proved to be sensitive, accurate and reliable for monitoring the emergence and early detection of HIV-1 genotypic variants that lead to drug resistance.
authors.affiliation: Research and Development Center, GeneMatrix Inc, Seongnam, South Korea.
authors.forename: J-H
authors.initials: JH
authors.lastname: Lee
authors.forename: A
authors.initials: A
authors.lastname: Hachiya
authors.forename: S-K
authors.initials: SK
authors.lastname: Shin
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: H
authors.initials: H
authors.lastname: Gatanaga
authors.forename: S
authors.initials: S
authors.lastname: Oka
authors.forename: K A
authors.initials: KA
authors.lastname: Kirby
authors.forename: Y T
authors.initials: YT
authors.lastname: Ong
authors.forename: S G
authors.initials: SG
authors.lastname: Sarafianos
authors.forename: W R
authors.initials: WR
authors.lastname: Folk
authors.forename: W
authors.initials: W
authors.lastname: Yoo
authors.forename: S P
authors.initials: SP
authors.lastname: Hong
authors.forename: S-O
authors.initials: SO
authors.lastname: Kim
mesh_terms: D000328:Adult; D000368:Aged; D019380:Anti-HIV Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D005838:Genotype; D015658:HIV Infections / Q000175:diagnosis / Q000188:drug therapy / Q000821:virology*; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019562:Viral Load; D055815:Young Adult
chemical_list: D019380:Anti-HIV Agents; D054303:HIV Reverse Transcriptase; D016333:HIV Protease