Format

Send to

Choose Destination
PLoS One. 2016 Dec 13;11(12):e0166902. doi: 10.1371/journal.pone.0166902. eCollection 2016.

An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies.

Author information

1
Centro de Biología Molecular 'Severo Ochoa' (CBMSO, CSIC-UAM). Campus de Cantoblanco, Madrid, Spain.
2
Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain.
3
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.
4
Department of Molecular Evolution, Centro de Astrobiología (CAB, CSIC-INTA). Torrejón de Ardoz, Madrid, Spain.
5
Biotherapix, SLU. Parque Tecnológico de Madrid, Tres Cantos, Madrid. Spain.
6
AIDS Immunopathogenesis Unit, Instituto de Salud Carlos III. Majadahonda, Madrid, Spain.

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.

PMID:
27959928
PMCID:
PMC5154500
DOI:
10.1371/journal.pone.0166902
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Competing Interests: We have the following interests: This study was supported by Biotherapix, S.L.U. Patricia Garrido, María Pernas and José Luis Torán were employed by Biotherapix, S.L.U. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center