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J Clin Microbiol. 2007 Aug;45(8):2604-15. Epub 2007 Jun 13.

Sensitive oligonucleotide ligation assay for low-level detection of nevirapine resistance mutations in human immunodeficiency virus type 1 quasispecies.

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  • 1Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Abstract

This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of approximately 0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population.

PMID:
17567789
[PubMed - indexed for MEDLINE]
PMCID:
PMC1951259
Free PMC Article
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