Development, evaluation, and validation of an oligonucleotide probe hybridization assay to subtype human immunodeficiency virus type 1 circulating recombinant form CRF02_AG

Abstract

We have developed and validated an oligonucleotide probe hybridization assay for human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) CRF02_AG. In the p17 coding region of the gag gene, a CRF02_AG-specific signature pattern was observed. Five working probes were designed to discriminate CRF02_AG infections from infections by all other documented subtypes and CRFs in an enzyme-linked immunosorbent assay-based oligonucleotide probe hybridization assay. Nucleic acids were extracted from a panel of HIV-1-positive plasma samples from Cameroon, Bénin, Côte d'Ivoire, Kenya, Zambia, and Belgium and from blood spots from The Gambia. CRF02_AG (n = 147) and non-CRF02 (n = 100) samples were analyzed to evaluate and validate the oligonucleotide probe hybridization assay. The CRF02_AG-specific oligonucleotide probe hybridization assay has a high sensitivity and specificity, with good positive and negative predictive values in regions of high and low prevalence. A validation of the assay with West and West Central African samples indicated a sensitivity of 98.4% and a specificity of 96.7%. The oligonucleotide probe hybridization assay as a diagnostic tool will allow for rapid screening for CRF02_AG. This could be used to track the HIV epidemic in terms of documenting the real prevalence of CRF02_AG strains and will complement efforts in vaccine development. Moreover, this technology can easily be applied in laboratories in developing countries.

FIG. 1.

Alignment of the conserved signature specific for CRF02_AG with all other subtypes and CRFs comprising fragments of subtype A. Black boxes indicate amino acid insertions and deletions within different subtypes and CRFs compared to probes (in bold and italics) specific for CRF02_AG.

FIG. 2.

OD distribution of evaluation panel samples. Oligonucleotide probe hybridization assay results are depicted according to genetic subtype classifications of CRF02_AG and non-CRF02_AG samples. A sample was considered to be CRF02_AG if it reacted with an OD value of >15.0 with either a probe or probe combination. Only the highest OD value obtained with probe combination PAg17α1 plus PAg17α2 or PAg17α3 plus PAg17α4 or with probe PAg17β is indicated.