Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout.

Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase.

Development and Optimization of Oligonucleotide Ligation Assay (OLA) Probes for Detection of HIV-1 Resistance to Dolutegravir.

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART.

Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma.

Background: Point-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow.

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.

A simple agglutination system for rapid antigen detection from large sample volumes with enhanced sensitivity.

Lateral flow assays (LFAs) provide a simple and quick option for diagnosis and are widely adopted for point-of-care or at-home tests. However, their sensitivity is often limited. Most LFAs only allow 50 μL samples while various sample types such as saliva could be collected in much larger volumes.

Implementation of an interactive mobile application to pilot a rapid assay to detect HIV drug resistance mutations in Kenya.

Usability is an overlooked aspect of implementing lab-based assays, particularly novel assays in low-resource-settings. Esoteric instructions can lead to irreproducible test results and patient harm. To address these issues, we developed a software application based on "Aquarium", a laboratory-operating system run on a computer tablet that provides step-by-step digital interactive instructions, protocol management, and sample tracking.

Patient-centered mobile tuberculosis treatment support tools (TB-TSTs) to improve treatment adherence: A pilot randomized controlled trial exploring feasibility, acceptability and refinement needs.

Background: Digital adherence technologies hold promise to improve patient-centered tuberculosis (TB) monitoring, yet few studies have incorporated adherence monitoring or assessed patients' experiences with these technologies. We explored acceptability, feasibility, and refinement needs of the TB Treatment Support Tools (TB-TSTs) intervention linking a mobile app, a urine drug metabolite test, and interactive communication with a treatment supporter.

Methods: This pilot study was a parallel-designed single-center randomized controlled trial with exit interviews.

Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes.

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control.

Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA).

The impact of a rapid home test on telehealth decision-making for influenza: a clinical vignette study.

Background: Home testing for influenza has the potential to aid triage and management decisions for patients with influenza-like illness. As yet, little is known about the effect of the home influenza testing on clinical decision-making via telehealth. The goal of this study was to determine the clinicians' decision thresholds for influenza and whether the availability of a home influenza test affects clinical decisions.

Diagnostic Accuracy of an At-Home, Rapid Self-test for Influenza: Prospective Comparative Accuracy Study.

Background: Rapid diagnostic tests (RDTs) for influenza used by individuals at home could potentially expand access to testing and reduce the impact of influenza on health systems. Improving access to testing could lead to earlier diagnosis following symptom onset, allowing more rapid interventions for those who test positive, including behavioral changes to minimize spread. However, the accuracy of RDTs for influenza has not been determined in self-testing populations.

Flu@home: the Comparative Accuracy of an At-Home Influenza Rapid Diagnostic Test Using a Prepositioned Test Kit, Mobile App, Mail-in Reference Sample, and Symptom-Based Testing Trigger.

At-home testing with rapid diagnostic tests (RDTs) for respiratory viruses could facilitate early diagnosis, guide patient care, and prevent transmission. Such RDTs are best used near the onset of illness when viral load is highest and clinical action will be most impactful, which may be achieved by at-home testing. We evaluated the diagnostic accuracy of the QuickVue Influenza A+B RDT in an at-home setting.

Isothermal Amplification with a Target-Mimicking Internal Control and Quantitative Lateral Flow Readout for Rapid HIV Viral Load Testing in Low-Resource Settings.

Point-of-care diagnostics often use isothermal nucleic acid amplification for qualitative detection of pathogens in low-resource healthcare settings but lack sufficient precision for quantitative applications such as HIV viral load monitoring. Although viral load (VL) monitoring is an essential component of HIV treatment, commercially available tests rely on relatively high-resource chemistries like real-time polymerase chain reaction and are thus used on an infrequent basis for millions of people living with HIV in low-income countries. To address the constraints of low-resource settings on nucleic acid quantification, we describe a recombinase polymerase amplification and lateral flow detection approach that quantifies HIV-1 DNA or RNA by comparison to a competitive internal amplification control (IAC) of a known copy number, which may be set to any useful threshold (in our case, a clinically relevant threshold for HIV treatment failure).

Requirements and Study Designs for U.S. Regulatory Approval of Influenza Home Tests.

Home testing for infectious disease has come to the forefront during the COVID-19 pandemic. There is now considerable commercial interest in developing complete home tests for a variety of viral and bacterial pathogens. However, the regulatory science around home infectious disease test approval and procedures that test manufacturers and laboratory professionals will need to follow have not yet been formalized by the U.

Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection.

RNA amplification tests sensitively detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but their complexity and cost are prohibitive for expanding coronavirus disease 2019 (COVID-19) testing. We developed “Harmony COVID-19,” a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device. Our ready-to-use, multiplexed reverse transcription, loop-mediated isothermal amplification (RT-LAMP) can detect down to 0.

A Systematic Review of Clinical Prediction Rules for the Diagnosis of Influenza.

Background: Clinical prediction rules (CPRs) can assist clinicians by focusing their clinical evaluation on the most important signs and symptoms, and if used properly can reduce the need for diagnostic testing. This study aims to perform an updated systematic review of clinical prediction rules and classification and regression tree (CART) models for the diagnosis of influenza.

Methods: We searched PubMed, CINAHL, and EMBASE databases.

Rapid Near Point-of-Care Assay for Genotype Associated with Severe Hypersensitivity Reaction to Abacavir.

The nucleoside reverse transcriptase inhibitor abacavir (ABC) is used commonly to treat young children with HIV infection and is a component of the fixed-dose-combination Triumeq. ABC can trigger a severe hypersensitivity reaction in people who are homozygous or heterozygous for . Testing for before ABC initiation is standard-of-care in high-resource settings, but current tests are costly or difficult to access in resource-limited settings.

Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing.

Background: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker.

Methods: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed.

Inexpensive workflow for simultaneous monitoring of HIV viral load and detection of SARS-CoV-2 infection.

Background: COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of becoming virologically unsuppressed and ill. Often they are at high risk for exposure to SARS-CoV-2 infection and severe disease once infected. For this population, it is urgent to closely monitor HIV plasma viral load ( ) and screen for SARS-COV-2 infection.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes.

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control.

Multivalent designed proteins protect against SARS-CoV-2 variants of concern.

Unlabelled: Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding.