CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

Scientific) and a published homebrew assay for integrase (Van Laethem 2008) (n=19) to assess assay accuracy. ONT sequence processing and drug resistance interpretation was with NanoRecall and the Stanford HIVdb 9.5.0. Results: PCR amplification success rates at viral load strata 200-1 000, 1 000-10 000 and >10 000 copies/mL were:15/28 (53.6%),15/20(75.0%) and 22/23(95.7%) respectively. Pairwise % identity between ONT and Sanger nucleotide sequences was 99.3% (98.3-100.0) for PR-RT and 99.4%(97.7-99.8) for IN; with a good agreement in major drug resistance mutations detected by both platforms (Figure). Conclusion: The novel fast ONT-based full pol-gene provides an alternative to Sanger Sequencing, especially in patients with higher viral loads. Discordant cases with Sanger Sequencing are likely a combination of PCR resampling error and small differences in the sequencing platforms. Our assay combined with NanoRecall interpretation provides an assay suitable for the use cases of drug resistance surveillance and individual patient management in resource limited settings.

utility in routine care. We previously developed the REverSe TRanscriptase Chain Termination (RESTRICT) assay to rapidly determine TFV-DP levels based on the drug's inhibition of DNA synthesis by HIV reverse transcriptase (RT). Here, we demonstrate the potential of a low-cost (<$300 USD) portable fluorometer, the Harmony reader, to provide semi-quantitative RESTRICT readout in near-patient settings. Methods: We validated semi-quantitative fluorescence measurements on the Harmony reader with RESTRICT assays containing DNA template, primers, nucleotides, recombinant HIV RT and TFV-DP. Reactions were incubated for 30 minutes at 37 °C, and PicoGreen intercalating dye was added to quench reactions and provide fluorescence readout. Harmony fluorescence measurements were compared to those obtained on a traditional benchtop plate reader (~$30,000 USD). Results: The Harmony reader (Fig 1A) integrates low-cost LEDs and sensors into a 3D-printed housing, is lightweight and portable, and can be USB powered. Harmony was able to detect TFV-DP in buffer at clinically relevant concentrations (Fig 1B). The resultant enzyme inhibition curve has a 50% inhibition concentration (IC 50 ) of 367 nM (95% confidence interval: 317-427 nM) corresponding to 750-1000 fmol/punch, demonstrating that the RESTRICT+Harmony platform can identify patients with TFV-DP concentrations that indicate moderate adherence (4 doses/week) . Harmony measurements were highly correlated with plate reader measurements (R 2 = 0.96, p<0.0001, N=120). Conclusion: Translation of RESTRICT fluorescent measurements to a portable reader eliminates reliance on expensive laboratory equipment and could enable deployment in near-patient settings (i.e. PrEP delivery settings, pharmacies, and clinics). Overall, RESTRICT+Harmony is rapid (<30 mins) and low-cost, and with the integration of sample preparation and a larger clinical validation, could accelerate global HIV prevention and treatment efforts by providing real-time DLF to support medication monitoring and adherence.

1100 A Rapid Emtricitabine Urine Lateral Flow Assay for Monitoring Daily Adherence to ART and PrEP Thomas H Vanderford 1 , Xierong Wei 2 , Ae S. Youngpairoj 2 , Ayanna Green 1 , Richard Haaland 1 , Jeffrey A. Johnson 2 , Walid Heneine 1 1 Centers for Disease Control and Prevention, Atlanta, GA, USA, 2 Centers for Disease Control and Prevention, Gaborone, Botswana Background: Adherence monitoring at point-of-care is challenging in the absence of rapid and accurate measures of antiretroviral drug levels. Emtricitabine (FTC) is a fixed dose component of most antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) regimens that is rapidly excreted in urine at high concentrations making it a suitable candidate for adherence measurement. We developed a test for daily adherence to FTC with a low-cost, rapid lateral flow assay (LFA) that detects FTC in urine. Here, we evaluate the performance of this LFA to discriminate daily adherence in people with HIV (PWH) on ART and individuals on daily PrEP. Methods: FTC-specific LFAs were designed and optimized in collaboration with a commercial manufacturer using a monoclonal antibody developed by our group. FTC LFA measurements were objectively read by a CubeReader (Chembio, Germany) with urine FTC concentrations >20 µg/mL indicating adherence to daily dosing. LFA performance (sensitivity and specificity) was evaluated on 273 urine samples which included (a) drug naïve, uninfected individuals (n = 62), (b) PWH prescribed ART (n = 67), and (c) people on PrEP (n = 144). FTC concentrations in urine and in plasma were measured by liquid chromatography tandem mass spectrometry for comparison with LFA results and to confirm adherence, respectively. Results: The FTC LFA provides results within 20 minutes. We evaluated the LFA for its ability to discriminate adherent from non-adherent individuals. FTC levels in plasma were used to stratify individuals for adherence based on a cutoff for daily adherence (>= 49 ng/mL plasma FTC concentration) optimized for greater than 90% sensitivity and specificity (Hendrix et al, 2016). The sensitivity of the LFA was 87.3% and its specificity was 93.5%. Thus, the FTC LFA performed well as a tool to determine daily adherence. Interestingly, median FTC levels in both plasma and urine were slightly higher (1.5-fold and 1.4-fold, respectively) in PWH on ART than in individuals on PrEP. Conclusion: We describe a lateral flow assay specific for FTC that is designed to detect whether a dose was taken within the past day. This daily adherence urine test has high sensitivity and specificity and can enhance point-of-care adherence monitoring for both ART and PrEP.

Poster Abstracts

1099 Full Pol-Gene PCR and Rapid ONT Library Preparation for Accurate Drug Resistance Sequencing Nicola Coetzee 1 , Conan K. Woods 2 , Kayla E. Delaney 1 , Mathilda Claassen 3 , Carli Gordijn 1 , Emma Sauermann 1 , Kim Steegen 4 , Urvi M. Parikh 5 , P Richard Harrigan 2 , Gert U. van Zyl 1 1 Stellenbosch University, Cape Town, South Africa, 2 University of British Columbia, Vancouver, Canada, 3 National Health Laboratory Service, Cape Town, South Africa, 4 University of the Witwatersrand, Johannesburg, South Africa, 5 University of Pittsburgh, Pittsburgh, PA, USA Background: Oxford Nanopore Technologies (ONT) third generation sequencing has the benefit of long read-length that allows the efficient and rapid sequencing of long DNA fragments. With the global rollout of integrase strand transfer inhibitors for treatment naïve and treatment experienced patients, drug resistance sequencing increasingly requires the sequencing of the protease (PR), reverse transcriptase (RT) and integrase (IN). We have therefore developed a rapid assay workflow that involves PCR amplification of a ~3 kb target, including all drug resistance mutations in pol, with rapid ONT barcode ligation followed by ONT real-time sequencing. Methods: HIV RNA was extracted with the NucliSens EasyMag (bioMeriuex) and amplified with a one-step reverse transcriptase PCR and nested PCR amplifying the ~3 kb PR-RT-IN target. Library preparation was with a fast ONT barcoding kit. We used residual HIV viral load samples (HIVVL) (n=71) to assess amplification success at various viral load strata. Residual samples from patients that had undergone Sanger Sequencing for protease and reverse transcriptase (n=35) with Applied Biosystems™ HIV-1 Genotyping Kit (ThermoFisher

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