HIV Pre-Exposure Prophylaxis Adherence Test Using Reverse Transcription Isothermal Amplification Inhibition Assay

Current HIV antiretroviral (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitoring drug dispensing, which are not reliable. We propose an objective adherence monitoring assay which directly measures nucleotide reverse transcriptase inhibitor (NRTI) concentration using a reverse transcription isothermal amplification inhibition assay. We measure the concentration of Tenofovir diphosphate (TFV-DP), a deoxyadenosine triphosphate (dATP) analog and an active NRTI compound and long-term adherence marker for PrEP, by measuring the inhibition of the reverse transcription of an RNA template. The completion or inhibition of reverse transcription is evaluated by Recombinase Polymerase Amplification (RPA), an isothermal nucleic acid amplification assay. We present and validate a model that predicts the amplification probability as function of dATP and TFV-DP concentrations, nucleotide binding sites on the RNA template, and the RNA template concentration. The model helps to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay can be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance.

On the Single-Crystal Structure of Tenofovir Alafenamide Mono-Fumarate: A Metastable Phase Featuring a Mixture of Co-Crystal and Salt

Tenofovir alafenamide (TAF) is a prodrug of tenofovir as a potent nucleotide reverse transcriptase inhibitor. It serves as the key component of Genvoya® for the first-line treatment of human immunodeficiency virus infection (HIV) and is the active component of Vemlidy® for the treatment of chronic hepatitis B. Vemlidy® is also a monotherapeutic regimen formulated as TAF hemifumarate (1; TAF:fumarate = 2:1). In this work, we report for the first time the single-crystal structure of TAF fumarate hemihydrate (2, TAF:fumarate:H2O = 2:2:1). Compound 2 is initially documented as a salt in which one proton of the fumaric acid migrates to the amine group of the adenine moiety in TAF. It was recently proposed that ca. 20–30% proton is transferred to the N atom on the aromatic adenine backbone. We herein provide definitive single-crystal X-ray diffraction results to confirm that 2, though phase pure, is formed as a mixture of co-crystal (75%) and salt (25%). It features two pairs of TAF fumarates, wherein one of the four H atoms on the fumaric acid is transferred to the N atom of the adjacent adenine moiety while the other three carboxylates remain in their intrinsic acid form. Compound 2 is a metastable phase during the preparation of 1 and can be isolated by halting the reaction during the refluxing of TAF and fumaric acid in acetonitrile (MeCN). Our report complements the previous characterizations of TAF monofumarate, and its elusive structural patterns are finally deciphered.

Diagnostic Accuracy of Pan-Degenerate Amplification and Adaptation Assay for HIV-1 Drug Resistance Mutation Analysis in Low- and Middle-Income Countries

ABSTRACT HIV drug resistance (HIVDR) is a barrier to sustained virologic suppression in low- and middle-income countries (LMICs). Point mutation assays targeting priority drug resistance mutations (DRMs) are being evaluated to improve access to HIVDR testing. In a cross-sectional study (June 2018 to September 2019), we evaluated the diagnostic accuracy of a simple and rapid HIVDR assay (the pan-degenerate amplification and adaptation [PANDAA] assay targeting the mutations K65R, K103NS, M184VI, Y181C, and G190A) compared to Sanger sequencing and next-generation sequencing (NGS). Plasma samples from adolescents and young adults (aged 10 to 24 years) failing antiretroviral therapy (viral load, >1,000 copies/ml on 2 consecutive occasions 1 month apart) were analyzed. Sensitivity and specificity of the PANDAA assay were determined by a proprietary application designed by Aldatu Biosciences. Agreement between genotyping methods was evaluated using Cohen’s kappa coefficient. One hundred fifty samples previously characterized by Sanger sequencing were evaluated using PANDAA. For all DRMs detected, PANDAA showed a sensitivity and specificity of 98% and 94%, respectively. For nucleotide reverse transcriptase inhibitor DRMs, sensitivity and specificity were 98% (95% confidence interval [CI], 92% to 100%) and 100% (94% to 100%), respectively. For non-nucleotide reverse transcriptase inhibitor DRMs, sensitivity and specificity were 100% (97% to 100%) and 76% (61% to 87%), respectively. PANDAA showed strong agreement with Sanger sequencing for K65R, K103NS, M184VI, and G190A (kappa > 0.85) and substantial agreement for Y181C (kappa = 0.720). Of the 21 false-positive samples genotyped by PANDAA, only 6 (29%) were identified as low-abundance variants by NGS. With the high sensitivity and specificity to detect major DRMs, PANDAA could represent a simple and rapid alternative HIVDR assay in LMICs.

Development of a Transdermal Delivery System for Tenofovir Alafenamide, a Prodrug of Tenofovir with Potent Antiviral Activity Against HIV and HBV

Tenofovir alafenamide (TAF) is an effective nucleotide reverse transcriptase inhibitor that is used in the treatment of HIV-1 and HBV. Currently, it is being investigated for HIV prophylaxis. Oral TAF regimens require daily intake, which hampers adherence and increases the possibility of viral resistance. Long-acting formulations would significantly reduce this problem. Therefore, the aim of this study was to develop a transdermal patch containing TAF and investigate its performance in vitro through human epidermis. Two types of TAF patches were manufactured. Transparent patches were prepared using acrylate adhesive (DURO-TAK 87-2516), and suspension patches were prepared using silicone (BIO-PSA 7-4301) and polyisobutylene (DURO-TAK 87-6908) adhesives. In vitro permeation studies were performed while using vertical Franz diffusion cells for seven days. An optimized silicone-based patch was characterized for its adhesive properties and tested for skin irritation. The acrylate-based patches, comprising 2% w/w TAF and a combination of chemical enhancers, showed a maximum flux of 0.60 ± 0.09 µg/cm2/h. However, the silicone-based patch comprising of 15% w/w TAF showed the highest permeation (7.24 ± 0.47 μg/cm2/h). This study demonstrates the feasibility of developing silicone-based transdermal patches that can deliver a therapeutically relevant dose of TAF for the control of HIV and HBV infections.