Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas.

Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25µL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.

REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention.

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in HIV care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase enzyme. Here, we introduce a probabilistic model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate, emtricitabine triphosphate, lamivudine triphosphate, and azidothymidine triphosphate with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring in HIV care and could be extended to other diseases treated with nucleotide analogs.

Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

HIV pre-exposure prophylaxis adherence test using reverse transcription isothermal amplification inhibition assay.

Current HIV antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitored drug dispensing, which are not reliable. We report a proof-of-concept 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) - an NRTI that functions as a deoxyadenosine triphosphate (dATP) analog 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 commonly used for point-of-care diagnostics. We present and validate a model that predicts the amplification probability as a function of dATP and TFV-DP concentrations, nucleotide insertion sites on the RNA template, and RNA template concentration. The model can be used to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay may be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance. Due to its simple instrumentation and short runtime (<1 hour), this assay has the potential for implementation in low-complexity laboratories or point-of-care settings, which may improve access to ART and PrEP adherence monitoring.

Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations.

OBJECTIVE: Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations-a metabolite that indicates long-term PrEP adherence. SETTING: The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle. METHODS: We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader-stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week). RESULTS: Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC-MS/MS. RESTRICT fluorescence correlated with LC-MS/MS measurements (r = - 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. CONCLUSIONS: This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

Pandemic-Related Instructor Talk: How New Instructors Supported Students at the Onset of the COVID-19 Pandemic.

At the same time that COVID-19 cases in the United States first began to increase, fellows in a mentored teaching apprenticeship for postdoctoral scientists began to teach undergraduate seminars. The fellows suddenly needed to support students emotionally and switch to online instruction. They were encouraged to acknowledge and address the pandemic during each class and decided to do so. In this case study, we examined the language fellows used in response to this encouragement, hypothesizing that they would engage in a variety of pandemic-related instructor talk, i.e., language that instructors use in the classroom that is not directly tied to educational content. We analyzed transcripts from 17 2-hour undergraduate biology seminar courses and found 167 instances of pandemic-related instructor talk. We used grounded theory to identify categories that emerged from these quotations: Positive coping mechanisms and self-care; Adjusting to online learning; Compassionate instruction; Personal impacts; COVID-19 and society; Dreaming; and Biology of COVID-19. Talk in these categories may help build relationships among instructors and students. The category about quickly Adjusting to online learning is unique, in that it is unlikely that there will be another time that will require simultaneous and rapid national movement to online instruction. In addition, four of the seven categories are direct consequences of COVID-19 specifically, and thus are unique to this time. Analyzing pandemic-related instructor talk has shed light on how new instructors navigated the trials of teaching in 2020.

Point-of-care and Near Real-time Testing for Antiretroviral Adherence Monitoring to HIV Treatment and Prevention.

PURPOSE OF REVIEW: In this report, we review the need for point-of-care (POC) or near real-time testing for antiretrovirals, progress in the field, evidence for guiding implementation of these tests globally, and future directions in objective antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) adherence monitoring. RECENT FINDINGS: Two cornerstones to end the HIV/AIDS pandemic are ART, which provides individual clinical benefits and eliminates forward transmission, and PrEP, which prevents HIV acquisition with high effectiveness. Maximizing the individual and public health benefits of these powerful biomedical tools requires high and sustained antiretroviral adherence. Routine monitoring of medication adherence in individuals receiving ART and PrEP may be an important component in interpreting outcomes and supporting optimal adherence. Existing practices and subjective metrics for adherence monitoring are often inaccurate or unreliable and, therefore, are generally ineffective for improving adherence. Laboratory measures of antiretroviral concentrations using liquid chromatography tandem mass spectrometry have been utilized in research settings to assess medication adherence, although these are too costly and resource-intensive for routine use. Newer, less costly technologies such as antibody-based methods can provide objective drug-level measurement and may allow for POC or near-patient adherence monitoring in clinical settings. When coupled with timely and targeted counseling, POC drug-level measures can support adherence clinic-based interventions to ART or PrEP in near real time.

Enzymatic Assay for Rapid Measurement of Antiretroviral Drug Levels.

Poor adherence to pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) can lead to human immunodeficiency virus (HIV) acquisition and emergence of drug-resistant infections, respectively. Measurement of antiviral drug levels provides objective adherence information that may help prevent adverse health outcomes. Gold-standard drug-level measurement by liquid chromatography/mass spectrometry is centralized, heavily instrumented, and expensive and is thus unsuitable and unavailable for routine use in clinical settings. We developed the REverSe TRanscrIptase Chain Termination (RESTRICT) assay as a rapid and accessible measurement of drug levels indicative of long-term adherence to PrEP and ART. The assay uses designer single-stranded DNA templates and intercalating fluorescent dyes to measure complementary DNA (cDNA) formation by reverse transcriptase in the presence of nucleotide reverse transcriptase inhibitor drugs. We optimized the RESTRICT assay using aqueous solutions of tenofovir diphosphate (TFV-DP), a metabolite that indicates long-term adherence to ART and PrEP, at concentrations over 2 orders of magnitude above and below the clinically relevant range. We used dilution in water as a simple sample preparation strategy to detect TFV-DP spiked into whole blood and accurately distinguished TFV-DP drug levels corresponding to low and high PrEP adherences. The RESTRICT assay is a fast and accessible test that could be useful for patients and clinicians to measure and improve ART and PrEP adherence.