title: Detection of HIV-1 antiretroviral resistance from patients with persistently low but detectable viraemia.
abstract: We modified the Abbott diagnostics HIV-1 Viroseq version 2 assay trade mark in order to detect the presence of HIV-1 drug resistance mutations in patients with viraemia below 1000 copies/ml of plasma. One hundred and forty-four patients with a detectable HIV-1 plasma viral load below 1000 copies/ml were selected and HIV-1 genetic analysis carried out using a modification of the Abbott Diagnostics Viroseq 2.0 assay trade mark. The procedure differs from the standard protocol in that a nested PCR amplification step was introduced. The oligonucleotide primers for the first round of PCR were those supplied in the RT-PCR module of the kit. The nested PCR primers were primers A and H taken from the sequencing module. One hundred and twenty-eight out of 144 (89%) plasma samples with an HIV-1 viral load of less than 1000 copies/ml (ranging from 54 to 992 copies) were successfully sequenced. HIV-1 genotypes were obtained from 68 out of 81 (84%) samples with a viral load of greater than 50 but less than 300 copies/ml and 60/63 (95%) of samples with a viral load of greater than 300 but less than 1000 copies/ml. Serial dilution of a sample with a high viral load did not affect the detection of resistance mutations. Multiple sequencing of samples with low viral load did not result in detection of additional mutations, although, in one sample the K103N mutation was detected in 3/6 replicates while wild-type was detected in 2/6 and a mixture of wild-type/mutant in 1/6. Samples from patients infected with both clade B and non-B clades of HIV-1 could be genotyped at low copy number. Modification of the Abbott Viroseq assay allows reproducible sequencing of the HIV-1 genome from patients with low, but detectable, plasma virus burden.
authors.affiliation: Division of Medicine, Wright Fleming Institute, Imperial College, St. Mary's Hospital, 4th Floor, Norfolk Palace, Paddington, London W2 1PG, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Mackie
authors.forename: Simon
authors.initials: S
authors.lastname: Dustan
authors.forename: Myra O
authors.initials: MO
authors.lastname: McClure
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Weber
authors.forename: John R
authors.initials: JR
authors.lastname: Clarke
mesh_terms: D019380:Anti-HIV Agents / Q000627:therapeutic use; D024882:Drug Resistance, Viral; D015751:Genes, pol; D005838:Genotype; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010802:Phylogeny; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D019562:Viral Load
chemical_list: D019380:Anti-HIV Agents; D012367:RNA, Viral