Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

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

Plate and paper formats for Capture, Denaturation and Detection (CDD) in the oligonucleotide ligation assay.

<p>(A) Plate CDD procedure. Products from the ligation step (both ligated and non-ligated products) are captured on a streptavidin-coated plate. Non-ligated probes are released during oligonucleotide denaturation, and ligated MUT and WT probes are then detected in sequential enzyme-based immunoassays (labeling by different detection antibodies, alkaline phosphatase yellow substrate development, optical density reading at 405nm, wash steps, tetramethylbenzidine (TMB) development, stop solution, and optical density reading at 450nm). (B) Paper CDD procedure. Similar to the plate CDD procedure, products from the ligation step (both ligated and non-ligated products) are captured. However, here the products are captured on paper strips by immobilized streptavidin. Non-ligated probes are released during oligonucleotide denaturation. Antibodies targeting the end-labels of the mutant (MUT) or wild-type (WT) probes have conjugated horseradish peroxidase (POD) that converts 3,3’ diazoaminobenzidine substrate (DAB) into brown precipitates. Signals were captured by the scanner (600 DPI). Reported signals represent capture spot intensity minus a background region from the strip.</p

Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations M184V and G190A.

<p>Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in pink and orange bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.</p

Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations K103N and Y181C.

<p>Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in blue and green bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.</p

Correlation plot of MUT Ratios determined by paper capture, denaturation, and detection (CDD) versus plate CDD.

<p>MUT and WT signals obtained by paper and plate CDD format OLA at codons K103N, Y181C, M184V and G190A from 26 clinical specimens were used to calculate MUT Ratios. The overall shape reflects the signal saturation of the paper CDD at high MUT concentration, as seen in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145962#pone.0145962.g003" target="_blank">3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145962#pone.0145962.g005" target="_blank">5</a>. Results correlate strongly across clinical specimens and plasmid standards’ data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145962#pone.0145962.s004" target="_blank">S4 Fig</a> for fitted correlation).</p

Comparison of paper Capture, Denaturation, and Detection (CDD) and plate CDD for analysis of a plasmid standard mixture series for mutation Y181C.

<p>(A) Scanned images of paper CDD MUT (left) and WT (right) detection (B) Mutant (MUT) detection (C) wild-type (WT) detection. Left axes: Specimen optical density (OD) minus negative control OD analyzed in duplicate by plate CDD (mean ± SE). Right axes: Specimen capture intensity minus background intensity analyzed in triplicate by paper CDD (mean ± SE). The ratio of mean signal intensities for 2% MUT and 0% MUT was 2.00 for plate CDD and 2.74 for paper CDD.</p

Schematic of amplification and ligation in the existing oligonucleotide ligation assay (OLA).

<p>The <i>pol</i> gene of HIV DNA is amplified by nested PCR. Note that the HIV DNA gene map positions are not to scale. A portion of the amplicon is mixed with three oligonucleotide probes: a 5’ fluorescein (F) -conjugated mutant (MUT)-specific HIV probe; a 5’ digoxigenin (D) -conjugated wild-type (WT)-specific probe; and a 5’ phosphorylated, 3’ biotin (B) -conjugated common probe. When specific probes are complementary at the mutation site, they are ligated to the common probe to create a DNA strand with labels at both ends. Only ligated products are detected during the CDD procedure (surface <u><b>c</b></u>apture, <u><b>d</b></u>enaturation of oligonucleotide from target DNA, and enzyme-based detection).</p

OLA-Simple : a software-guided HIV-1 drug resistance test for low-resource laboratories

Background: HIV drug resistance (HIVDR) testing can assist clinicians in selecting treatments. However, high complexity and cost of genotyping assays limit routine testing in settings where HIVDR prevalence has reached high levels. Methods: The oligonucleotide ligation assay (OLA)-Simple kit was developed for detection of HIVDR against first-line non-nucleoside/nucleoside reverse transcriptase inhibitors and validated on 672 codons (168 spedmens) from subtypes A, B, C, D, and AE. The kit uses dry reagents to facilitate assay setup, lateral flow devices for visual HIVDR detections, and in-house software with an interface for guiding users and analyzing results. Findings: HIVDR analysis of specimens by OLA-Simple compared to Sanger sequencing revealed 99.6 +/- 0.3% specificity and 98.2 +/- 0.9% sensitivity, and compared to high-sensitivity assays, 99.6 +/- 0.6% specificity and 86.2 +/- 2.5% sensitivity, with 2.6 +/- 0.9% indeterminate results. OLA-Simple was performed more rapidly compared to Sanger sequencing (<4 h vs. 35-72 h). Forty-one untrained volunteers blindly tested two specimens each with 96.8 +/- 0.8% accuracy. Interpretation: OLA-Simple compares favorably with HIVDR genotyping by Sanger and sensitive comparators. Instructional software enabled inexperienced, first-time users to perform the assay with high accuracy. The reduced complexity, cost, and training requirements of OLA-Simple could improve access to HIVDR testing in low-resource settings and potentially allow same-day selection of appropriate antiretroviral therapy