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Global Health Technologies

Global Health Technologies - Poster Session D

Poster Y5 - Detection of HIV-1 Resistance to Dolutegravir by an Oligonucleotide Ligation Assay using Lateral Flow Strips

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  In 2019, the World Health Organization recommended Dolutegravir (DTG)-based regimens as the preferred first-line antiretroviral therapy (ART) due to randomized trials showing the superiority of DTG-based-ART compared to non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens, and increased prevalence of HIV drug resistance (HIVDR) to the NNRTI’s. However, clinics report that 3.9%-19.6% of individuals on DTG-based regimens have experienced unsuppressed viremias.

Access to point-of-care diagnostics for the timely detection of HIVDR mutations to DTG would allow clinicians to determine if changes in ART or adherence counseling is needed. This is particularly crucial in low-resource settings, where the common detection methods for HIVDR, such as Sanger Sequencing or Next Generation Sequencing, are inaccessible due to cost and infrastructure limitations.

To address this need, we have adapted the OLA-Simple assay to detect HIVDR to DTG. OLA-Simple is a cost-effective, rapid, and user-friendly oligonucleotide ligation assay. It was previously developed to detect HIVDR mutations against NNRTI and nucleoside reverse transcriptase inhibitors. The OLA-Simple assay involves four steps: sample preparation, target amplification, target-specific ligation of genotype-specific probes, and colorimetric detection of results via a lateral flow test.

In this work, OLA-Simple was developed to detect G118R, Q148H/K/R, N155H, and R263K mutations in the HIV integrase gene in HIV-1 subtypes A, B, C, D, and CRF01_AE. These mutations have been identified as the most prevalent single mutations and represent non-overlapping mutational pathways.

Materials and

Methods:  Standard mixtures containing 0%, 10%, 15%, 25%, 50%, or 100% mutant (MUT) plasmid mixed with wildtype (WT) plasmid were used as sample input. These plasmid mixtures were amplified using real-time PCR, and melt curve analysis was used to assess PCR performance. Post-PCR, the amplicon was added to six different ligation reactions containing ligase enzyme, its cofactors, and probes for detecting G118R, Q148H/K/R, N155H, and R263K, respectively, as described. The probe mixture contains 5’fluorescein-labeled MUT probe, 5’digoxigenin- labeled WT probe, and 5’phosphorylated 3’biotin-labeled common (COM) probe. Post-ligation, unlabeled competing oligos for each mutation were added to the corresponding ligation reactions, and a single cycle of ligation was performed to displace the target amplicons from the ligation products and thus minimize false signals. The ligation mixture was then loaded onto the sample port of a lateral flow strip immobilized with biotin-BSA as the control line, anti-digoxigenin IgG as the WT line, and anti-fluorescein IgG as the MUT line. Gold nanoparticle solution was applied after a 5-minute wait. Subsequently, the strips were scanned after 15 and 20 minutes. Quantification of the signal was performed using ImageJ (blue channel) by subtracting background region intensity from the band intensity region. Analyses from two operators were averaged to report the final intensity and plotted along with standard deviations (SD).

Results, Conclusions, and Discussions: Among the DTG integrase mutations tested here, we observed expected results with visible differences between 0% and 10% MUT band intensities in R263K, N155H, Q148K, and Q148H, which are in concordance with software analysis (Fig. 1). For Q148H, an unexpected background MUT line was observed in the 0% MUT sample, yet there were visible differences in the intensities between the 0% and 10% MUT mixtures. This background MUT signal could be attributed to an excessively high ligase concentration, allowing off-target ligation. In contrast, the G118R reactions produced visible MUT lines only for samples with more than 50% MUT, and this could potentially be optimized by increasing the MUT ligation probe concentration. Q148R standard mixtures generated visible MUT and WT lines in all concentrations, including no template controls. For Q148R, we will confirm the identity of our MUT and WT plasmid stocks by sequencing. In conclusion, this work serves as the first step towards the development of OLA-Simple for the rapid detection of DTG HIVDR mutations, which has the potential to offer affordable and accessible detection of DTG HIVDR mutations in resource-limited settings. Future research will focus on optimizing the assay conditions for improved lateral flow detection and sensitivity at codons G118R, Q148H, and Q148R.

Acknowledgements (Optional):  We thank Lutz lab members Dr. Qin Wang and Shane Gilligan-Steinberg for providing training on lateral flow test preparation and Panpradist lab member Inyoung Seo for providing training on other data analysis methods. This work was funded by the NIH R01AI145486 (PI: Lutz) and the 2022 SEATRAC New Investigator Award (PI: Dr. Panpradist). The funders have no role in study design, experimental results, and interpretations.