An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies
Martín, Verónica (Centro de Biología Molecular Severo Ochoa)
Perales Viejo, Celia (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Fernández-Algar, María (Centro de Astrobiología (Madrid))
Dos Santos, Helena G. (Centro de Biología Molecular Severo Ochoa)
Garrido, Patricia (Biotherapix (Madrid))
Pernas, María (Biotherapix (Madrid))
Parro, Víctor (Centro de Astrobiología (Madrid))
Moreno, Miguel (Centro de Astrobiología (Madrid))
García-Pérez, Javier (Instituto de Salud Carlos III)
Alcami, Jose (Instituto de Salud Carlos III)
Torán, José Luis (Biotherapix (Madrid))
Abia, David (Centro de Biología Molecular Severo Ochoa)
Domingo, Esteban (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas)
Briones Llorente, Carlos (Centro de Astrobiología (Madrid))
Universitat Autònoma de Barcelona

Data:	2016
Resum:	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.
Ajuts:	Ministerio de Ciencia e Innovación BIO2010-20696 Ministerio de Economía y Competitividad BIO2013-47228-R Ministerio de Economía y Competitividad SAF2014-52400-R Ministerio de Ciencia e Innovación RyC2010-06516 Ministerio de Ciencia e Innovación AGL2015-64290-R Instituto de Salud Carlos III CP14/00121
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Publicat a:	PloS one, Vol. 11 (december 2016) , ISSN 1932-6203

DOI: 10.1371/journal.pone.0166902
PMID: 27959928

24 p, 2.3 MB

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