title: Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.
abstract: Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Nuttada
authors.initials: N
authors.lastname: Panpradist
authors.affiliation: Seattle Children's Research Institute, Seattle, Washington, United States of America.
authors.forename: Ingrid A
authors.initials: IA
authors.lastname: Beck
authors.affiliation: Department of Internal Medicine, University of Washington, Seattle, Washington, United States of America.|Department of Global Health, University of Washington, Seattle, Washington, United States of America.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Chung
authors.affiliation: Departments of Obstetrics and Gynecology, University of Nairobi, Nairobi, Kenya.
authors.forename: James N
authors.initials: JN
authors.lastname: Kiarie
authors.affiliation: Department of Global Health, University of Washington, Seattle, Washington, United States of America.|Departments of Pediatrics and Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Frenkel
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, United States of America.
authors.forename: Barry R
authors.initials: BR
authors.lastname: Lutz
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D059788:Dried Blood Spot Testing / Q000191:economics / Q000379:methods; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques; D015658:HIV Infections / Q000175:diagnosis / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007630:Kenya; D022521:Ligase Chain Reaction / Q000191:economics / Q000379:methods*; D025202:Molecular Diagnostic Techniques; D015345:Oligonucleotide Probes / Q000737:chemistry; D010209:Paper; D017354:Point Mutation; D012189:Retrospective Studies; D059629:Signal-To-Noise Ratio; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D019380:Anti-HIV Agents; D015345:Oligonucleotide Probes; D054302:pol Gene Products, Human Immunodeficiency Virus