title: An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies.
abstract: The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa' (CBMSO, CSIC-UAM). Campus de Cantoblanco, Madrid, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Martín
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa' (CBMSO, CSIC-UAM). Campus de Cantoblanco, Madrid, Spain.|Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain.|Liver Unit, Internal Medicine, Laboratory of Malalties Hepàtiques, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d´Hebron (VHIR-HUVH), Universitat Autònoma de Barcelona. Barcelona, Spain.
authors.forename: Celia
authors.initials: C
authors.lastname: Perales
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CAB, CSIC-INTA). Torrejón de Ardoz, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Fernández-Algar
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa' (CBMSO, CSIC-UAM). Campus de Cantoblanco, Madrid, Spain.
authors.forename: Helena G
authors.initials: HG
authors.lastname: Dos Santos
authors.affiliation: Biotherapix, SLU. Parque Tecnológico de Madrid, Tres Cantos, Madrid. Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Garrido
authors.affiliation: Biotherapix, SLU. Parque Tecnológico de Madrid, Tres Cantos, Madrid. Spain.
authors.forename: María
authors.initials: M
authors.lastname: Pernas
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CAB, CSIC-INTA). Torrejón de Ardoz, Madrid, Spain.
authors.forename: Víctor
authors.initials: V
authors.lastname: Parro
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CAB, CSIC-INTA). Torrejón de Ardoz, Madrid, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Moreno
authors.affiliation: AIDS Immunopathogenesis Unit, Instituto de Salud Carlos III. Majadahonda, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: García-Pérez
authors.affiliation: AIDS Immunopathogenesis Unit, Instituto de Salud Carlos III. Majadahonda, Madrid, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Alcamí
authors.affiliation: Biotherapix, SLU. Parque Tecnológico de Madrid, Tres Cantos, Madrid. Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Torán
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa' (CBMSO, CSIC-UAM). Campus de Cantoblanco, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Abia
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa' (CBMSO, CSIC-UAM). Campus de Cantoblanco, Madrid, Spain.|Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain.
authors.forename: Esteban
authors.initials: E
authors.lastname: Domingo
authors.affiliation: Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain.|Department of Molecular Evolution, Centro de Astrobiología (CAB, CSIC-INTA). Torrejón de Ardoz, Madrid, Spain.
authors.forename: Carlos
authors.identifier: http://orcid.org/0000-0003-2213-8353
authors.initials: C
authors.lastname: Briones
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods* / Q000592:standards; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards
chemical_list: D019380:Anti-HIV Agents