Suchergebnisse

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. ; This work was supported by the Spanish Ministerio de Ciencia e Innovación (MICINN grant BIO2010-20696 to CB), Ministerio de Economía y Competitividad (MINECO grants BIO2013-47228-R to CB and SAF2014-52400-R to ED), Comunidad Autónoma de Madrid (PLATESA, grant S2013/ABI-2906 to ED), Biotherapix, S.L.U. and FEDER funds from the European Union. Work at CIBERehd is funded by the Instituto de Salud Carlos III (ISCIII). MICINN provided funding for VM under grants RyC2010-06516 and AGL2015-64290-R. CP is supported by Miguel Servet program of the ISCIII (CP14/00121), co-financed by the European Regional Development Fund (ERDF). Funding for open access charge: Spanish National Research Council (CSIC). The funders provided support in the form of salaries and research materials for authors [MICINN and MINECO for VM, CP, MF-A, HGDS, VP, MM, DA, ED and CB; ISCIII for CP, JG-P and JA; Biotherapix, S.L.U. for PG, MP and JLT], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section. ; Sí