title: A genotypic assay for the amplification and sequencing of integrase from diverse HIV-1 group M subtypes.
abstract: Recently, the Food and Drug Administration (FDA) of the USA approved the first integrase inhibitor for inclusion in treatment regimens of HIV-1 patients failing their current regimens with multi-drug resistant strains. However, treatment failure has been observed during integrase inhibitor-containing therapy. Several mutational pathways have been described with signature mutations at integrase positions 66, 92, 148 and 155. Therefore, a genotypic assay for the amplification and sequencing of HIV-1 integrase was developed. The assay displayed a detection limit of 10 HIV-1 III(B) RNA copies/ml plasma. As the HIV-1 pandemic is characterised by a large genetic diversity, the new assay was evaluated on a panel of 74 genetically divergent samples belonging to the following genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRFF03-AB, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 until >500,000 RNA copies/ml. The amplification and sequencing was successful for 70 samples (a success rate of 95%). The four failures were most probably due to low viral load or poor quality of RNA and not to subtype issues. Some of the sequences obtained from integrase inhibitor-naïve patients displayed polymorphisms at integrase positions associated with resistance: 74IV, 138D, 151I, 157Q and 163AE. The relevance of these polymorphisms in the absence of the signature mutations remains unclear.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. kristel.vanlaethem@uz.kuleuven.ac.be
authors.forename: Kristel
authors.initials: K
authors.lastname: Van Laethem
authors.forename: Yoeri
authors.initials: Y
authors.lastname: Schrooten
authors.forename: Kris
authors.initials: K
authors.lastname: Covens
authors.forename: Nathalie
authors.initials: N
authors.lastname: Dekeersmaeker
authors.forename: Paul
authors.initials: P
authors.lastname: De Munter
authors.forename: Eric
authors.initials: E
authors.lastname: Van Wijngaerden
authors.forename: Marc
authors.initials: M
authors.lastname: Van Ranst
authors.forename: Anne-Mieke
authors.initials: AM
authors.lastname: Vandamme
mesh_terms: D018076:DNA, Complementary; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D019427:HIV Integrase / Q000235:genetics*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D011760:Pyrrolidinones / Q000494:pharmacology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D000068898:Raltegravir Potassium; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D019428:HIV Integrase Inhibitors; D011760:Pyrrolidinones; D012367:RNA, Viral; D000068898:Raltegravir Potassium; D019427:HIV Integrase