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.

The evolution of HIV‐1 group M genetic variability in Southern Cameroon is characterized by several emerging recombinant forms of CRF02_AG and viruses with drug resistance mutations

“…Some viral variants may have higher replicative fitness or transmissibility or may be dominant in a population due to founder effect. For example, the predominance of CRF02_AG suggests that this virus strain may be well adapted in the Cameroonian population due to a founder effect [Njai et al, 2004] or has some biologic advantages such as a higher replicative fitness and modification of tropism over other cocirculating strains [Montavon et al, 2000;Fischetti et al, 2004;Sarr et al, 2005;Konings et al, 2006a;Njai et al, 2006]. In this study, (sub)subtypes that were concordant in all four genetic regions analyzed, referred to here as "pure" subtypes, were detected with low prevalence and accounted for only 3/111 (2.7%) of infections.…”

Oligonucleotide Ligation Assay for Detection of Mutations Associated with Reverse Transcriptase and Protease Inhibitor Resistance in Non-B Subtypes and Recombinant Forms of Human Immunodeficiency Virus Type 1

“…In contrast to the LiPA assay, the OLA is highly adaptable since it allows the design of novel and specific oligonucleotides to detect new resistanceassociated mutations as they are identified or to detect resistance-associated mutations in non-B subtype samples, where multiple genetic polymorphisms and accessory mutations are commonly found (1,7,16). This assay could also be used for the efficient determination and discrimination of different genetic subtypes through the design of specific oligonucleotides, as previously described for a similar genotyping approach (10,12). Moreover, the OLA has the ability to detect drug-resistant subpopulations present in a relatively small proportion (8), which represents a big advantage compared to sequencing because this makes OLA useful for the election of initial therapeutic regimens or changes in therapy in order to prevent the accumulation of additional mutations.…”