Search

The current four symptom screen recommended by the WHO is widely used as screen to initiate diagnostic testing for active pulmonary tuberculosis (TB), yet the performance is poor especially when TB prevalence is low. In contrast, more sensitive molecular tests are less suitable for the placement at primary care level in low resource settings. In order to meet the WHO End TB targets new diagnostic approaches are urgently needed to find the missing undiagnosed cases. Proteomics-derived blood host biomarkers have been explored because protein detection technologies are suitable for the point-of-care setting and could meet cost targets.This study aims to find a biomarker signature that fulfills WHO’s target product profile (TPP) for a TB screening. 12 blood-based protein biomarkers from three sample populations (Vietnam, Peru, South Africa) are analyzed individually and in combinations via advanced statistical methods and machine learning algorithms. The combination of I-309, SYWC and kallistatin shows the most promising results for TB prediction throughout the datasets meeting the TPP for a triage test in adults from two countries (Peru and South Africa). The top performing individual markers identified at the global level (I-309 and SYWC) were also among the best performing markers at country level in South Africa and Vietnam.This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions based on one or two biomarkers with a performance that meets minimal WHO TPP criteria.Abstract ImportanceTuberculosis (TB) remains a serious worldwide health problem and diagnosis is hampered by the complexity of tests at primary care level in low resource settings or the low accuracy for screening settings. In order to meet the WHO End TB targets new diagnostic approaches are urgently needed to find the missing undiagnosed cases.This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions. We were able to construct an algorithm through statistical methods and machine learning algorithms whose performance meets the minimum of the WHO target product profile criteria. Thus, further work should be addressed at demonstrating that such as assay can be translated into a practical point-of-care test, and to better understand how to address regional differences in biomarker levels and responses.

Poor patient adherence to antiretroviral medication represents a major obstacle for managing disease and reducing rates of new HIV infections. The measurement of patient drug levels is the most objective method of determining adherence. Tenofovir and tenofovir diphosphate are metabolites of some of the most common HIV medications for treatment and prevention and can be quantified by mass spectrometry. Here, we report the development of a competitive enzyme linked immunoassay as a simplified approach for detecting tenofovir and tenofovir diphosphate. Monoclonal antibodies were produced by two tenofovir-hapten conjugates and screened for binding to immobilized tenofovir, and then for competition by tenofovir and tenofovir diphosphate. Antibody specificity was evaluated against adenosine phosphates, which are close structural analogs. We performed numerical simulations of reaction equilibrium to guide assay optimization. When used to evaluate spiked tenofovir in plasma and spiked tenofovir diphosphate in red blood cell lysate, the optimized assay had high sensitivity and specificity.

Background: HIV self-testing (HIVST) is recommended by the WHO as an innovative strategy to reach UNAIDS targets to end HIV by 2030. HIVST with digital supports is defined as the use of digital interventions (e.g., website-based, social media, mobile HIVST applications (apps), text messaging (SMS), digital vending machines (digital VMs)) to improve the efficiency and impact of HIVST. HIVST deployment and integration in health services is an emerging priority. We conducted a systematic review aiming to close the gap in evidence that summarizes the impact of digitally supported HIVST and to inform policy recommendations. Methods: We searched PubMed and Embase for articles and abstracts on HIVST with digital supports published during the period February 1st, 2010 to June 15th, 2021, following Cochrane guidelines and PRISMA methodology. We assessed feasibility, acceptability, preference, and impact outcomes across all populations and study designs. Metrics reported were willingness to use HIVST, preferences for HIVST delivery, proportion of first-time testers, HIVST uptake, HIVST kit return rate, and linkage to care. Heterogeneity of the interventions and reported metrics precluded us from conducting a meta-analysis. Findings: 46 studies were narratively synthesized, of which 72% were observational and 28% were RCTs. Half of all studies (54%, 25/46) assessed web-based innovations (e.g., study websites, videos, chatbots), followed by social media (26%, 12/46), HIVST-specific apps (7%, 3/46), SMS (9%, 4/46), and digital VMs (4%, 2/46). Web based innovations were found to be acceptable (77-97%), preferred over in-person and hybrid options by more first-time testers (47-48%), highly feasible (93-95%), and were overall effective in supporting linkage to care (53-100%). Social media and app-based innovations also had high acceptability (87-95%) and linkage to care proportions (80-100%). SMS innovations increased kit return rates (54-94%) and HIVST uptake among hard-to-reach groups. Finally, digital VMs were highly acceptable (54-93%), and HIVST uptake was six times greater when using digital VMs compared to distribution by community workers. Interpretation: HIVST with digital supports was deemed feasible, acceptable, preferable, and was shown to increase uptake, engage first-time testers and hard-to-reach populations, and successfully link participants to treatment. Findings pave the way for greater use of HIVST interventions with digital supports globally. Funding: This work was funded by the Foundation for Innovative New Diagnostics. The agency had no role in the decision to submit it for publication, however the funders contributing to the writing of the manuscript. NPP also acknowledges support from the Fonds de recherche du Quebec Sante (Senior scientist scholar award), The Canadian Institutes of Health Research (PJT 153149 and HBR 422155), Grand Challenges Canada (Transition to Scale award, 071005), the India-Canada centre for Innovative Multidisciplinary Partnerships to Accelerate Community Transformation and Sustainability (IC-IMPACTS), and the MUHC Foundation. (c) 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 two 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 adherence. The RESTRICT assay is a fast and accessible test that could be useful test for patients and clinicians to measure and improve ART and PrEP adherence.

Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains., Author summary Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a serious global health problem, causing ~1.5 million deaths a year world-wide. Like other bacterial pathogens, diversity among strains of Mtb contributes to differences in infection outcomes, vaccine efficacy, and response to antibiotic treatment. Currently, the important genetic differences that contribute to variation among Mtb strains remain poorly understood. In this study, we applied a functional genomics technique called TnSeq to a panel of Mtb clinical strains to investigate the genetic basis of strain diversity. We identified a number of genes that are differentially required for growth in culture among these strains. Some of these genes are involved in the response to antibiotics, including the first-line antitubercular agent isoniazid and a novel antitubercular drug currently in development. We found that the genetic differences between strains uncovered by TnSeq predicted responses to these antibiotics. Our results demonstrate the utility of TnSeq for identifying clinically relevant differences among Mtb strains.

The development of faster and more sensitive detection methods capable of identifying specific bacterial species and strains has remained a longstanding clinical challenge. Thus to date, the diagnosis of bacterial infections continues to rely on the performance of time-consuming microbiological cultures. Here, we demonstrate the use of bioorthogonal chemistry for magnetically labeling specific pathogens to enable their subsequent detection by nuclear magnetic resonance. Antibodies against a bacterial target of interest were first modified with trans-cyclooctene and then coupled to tetrazine-modified magnetic nanoprobes, directly on the bacteria. This labeling method was verified by surface plasmon resonance as well as by highly specific detection of Staphylococcus aureus using a miniaturized diagnostic magnetic resonance system. Compared to other copper-free bioorthogonal chemistries, the cycloaddition reaction reported here displayed faster kinetics and yielded higher labeling efficiency. Considering the short assay times and the portability of the necessary instrumentation, it is feasible that this approach could be adapted for clinical use in resource-limited settings.

The task of rapidly identifying patients infected with Mycobacterium tuberculosis in resource-constrained environments remains a challenge. A sensitive and robust platform that does not require bacterial isolation or culture is critical in making informed diagnostic and therapeutic decisions. Here we introduce a platform for the detection of nucleic acids based on a magnetic barcoding strategy. PCR-amplified mycobacterial genes are sequence-specifically captured on microspheres, labelled by magnetic nanoprobes and detected by nuclear magnetic resonance. All components are integrated into a single, small fluidic cartridge for streamlined on-chip operation. We use this platform to detect M. tuberculosis and identify drug-resistance strains from mechanically processed sputum samples within 2.5 h. The specificity of the assay is confirmed by detecting a panel of clinically relevant non-M. tuberculosis bacteria, and the clinical utility is demonstrated by the measurements in M. tuberculosis-positive patient specimens. Combined with portable systems, the magnetic barcode assay holds promise to become a sensitive, high-throughput and low-cost platform for point-of-care diagnostics.

Author Manuscript 2011 July 22, Solid materials, such as silicon, glass, and polymers, dominate as structural elements in microsystems including microfluidics. Porous elements have been limited to membranes sandwiched between microchannel layers or polymer monoliths. This paper reports the use of micropatterned carbon-nanotube forests confined inside microfluidic channels for mechanically and/or chemically capturing particles ranging over three orders of magnitude in size. Nanoparticles below the internanotube spacing (80 nm) of the forest can penetrate inside the forest and interact with the large surface area created by individual nanotubes. For larger particles (>80 nm), the ultrahigh porosity of the nanotube elements reduces the fluid boundary layer and enhances particle–structure interactions on the outer surface of the patterned nanoporous elements. Specific biomolecular recognition is demonstrated using cells (≈10 μm), bacteria (≈1 μm), and viral-sized particles (≈40 nm) using both effects. This technology can provide unprecedented control of bioseparation processes to access bioparticles of interest, opening new pathways for both research and point-of-care diagnostics., National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant P41 EB002503), United States. Department of State (Fulbright Science and Technology Award)

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

Recent advances in fluorescent probe technology have improved spatial and temporal resolution, bringing us closer to the ideal of imaging individual cellular features in real time with molecular (1–5 nm) resolution. In parallel, the development of super-resolution imaging techniques has revolutionized fluorescence microscopy. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1–5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

We report a new method for detection of protein-protein interactions in vitro and in cells. One protein partner is fused to Escherichia coli biotin ligase (BirA), while the other protein partner is fused to BirA's "acceptor peptide" (AP) substrate. If the two proteins interact, BirA will catalyze site-specific biotinylation of AP, which can be detected by streptavidin staining. To minimize nonspecific signals, we engineered the AP sequence to reduce its intrinsic affinity for BirA. The rapamycin-controlled interaction between FKBP and FRB proteins could be detected in vitro and in cells with a signal to background ratio as high as 28. We also extended the method to imaging of the phosphorylation-dependent interaction between Cdc25C phosphatase and 14-3-3epsilon phosphoserine/threonine binding protein. Protein-protein interaction detection by proximity biotinylation has the advantages of low background, high sensitivity, small AP tag size, and good spatial resolution in cells.