Molecular point of care testing for hepatitis C: available technologies, pipeline and promising future directions.

Hepatitis C (HCV) remains a major public health problem, despite the availability of effective treatments. In many areas, the ability to diagnose HCV infection at the point of care is key to scaling up access to care and treatment. To achieve this, an accurate, easy-to-use and affordable diagnostic tool is required - this would enable decentralized testing and the creation of one-stop centers to eliminate gaps in the care cascade, which would help reach the millions of people with undiagnosed HCV in low- and middle-income countries and high-risk populations in high income countries. In this review, we examine the current state of point-of-care molecular technologies, the advantages and limitations of currently available devices (both near- and true-point-of-care), the potential of molecular testing to transform diagnostic medicine in the future, and the challenges that need to be addressed for broader adoption of this technology in routine clinical practice.

Diagnostic performance of host protein signatures as a triage test for active pulmonary TB.

The current four-symptom screen recommended by the World Health Organization (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 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 aimed to find a biomarker signature that fulfills WHO's target product profile (TPP) for a TB screening. Twelve blood-based protein biomarkers from three sample populations (Vietnam, Peru, and South Africa) were analyzed individually and in combinations via advanced statistical methods and machine learning algorithms. The combination of I-309, SYWC and kallistatin showed the most promising results to discern active TB throughout the data sets 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.

Self-sampling strategies (with/without digital innovations) in populations at risk of Chlamydia trachomatis and Neisseria gonorrhoeae: a systematic review and meta-analyses.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) resulted in over 200 million new sexually transmitted infections last year. Self-sampling strategies alone or combined with digital innovations (ie, online, mobile or computing technologies supporting self-sampling) could improve screening methods. Evidence on all outcomes has not yet been synthesised, so we conducted a systematic review and meta-analysis to address this limitation. METHODS: We searched three databases (period: 1 January 2000-6 January 2023) for reports on self-sampling for CT/GC testing. Outcomes considered for inclusion were: accuracy, feasibility, patient-centred and impact (ie, changes in linkage to care, first-time testers, uptake, turnaround time or referrals attributable to self-sampling).We used bivariate regression models to meta-analyse accuracy measures from self-sampled CT/GC tests and obtain pooled sensitivity/specificity estimates. We assessed quality with Cochrane Risk of Bias Tool-2, Newcastle-Ottawa Scale and Quality Assessment of Diagnostic Accuracy Studies-2 tool. RESULTS: We summarised results from 45 studies reporting self-sampling alone (73.3%; 33 of 45) or combined with digital innovations (26.7%; 12 of 45) conducted in 10 high-income (HICs; n=34) and 8 low/middle-income countries (LMICs; n=11). 95.6% (43 of 45) were observational, while 4.4% (2 of 45) were randomised clinical trials.We noted that pooled sensitivity (n=13) for CT/GC was higher in extragenital self-sampling (>91.6% (86.0%-95.1%)) than in vaginal self-sampling (79.6% (62.1%-90.3%)), while pooled specificity remained high (>99.0% (98.2%-99.5%)).Participants found self-sampling highly acceptable (80.0%-100.0%; n=24), but preference varied (23.1%-83.0%; n=16).Self-sampling reached 51.0%-70.0% (n=3) of first-time testers and resulted in 89.0%-100.0% (n=3) linkages to care. Digital innovations led to 65.0%-92% engagement and 43.8%-57.1% kit return rates (n=3).Quality of studies varied. DISCUSSION: Self-sampling had mixed sensitivity, reached first-time testers and was accepted with high linkages to care. We recommend self-sampling for CT/GC in HICs but additional evaluations in LMICs. Digital innovations impacted engagement and may reduce disease burden in hard-to-reach populations. PROSPERO REGISTRATION NUMBER: CRD42021262950.

Immunoassay for HIV Drug Metabolites Tenofovir and Tenofovir Diphosphate.

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.

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.

Correction: TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities.

[This corrects the article DOI: 10.1371/journal.ppat.1006939.].

TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities.

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.

Magnetic barcode assay for genetic detection of pathogens.

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.

Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria.

The ESX-1 secretion system is required for pathogenicity of Mycobacterium tuberculosis (Mtb). Despite considerable research, little is known about the structural components of ESX-1, or how these proteins are assembled into the active secretion apparatus. Here, we exploit the functionally related ESX-1 apparatus of Mycobacterium smegmatis (Ms) to show that fluorescently tagged proteins required for ESX-1 activity consistently localize to the cell pole, identified by time-lapse fluoro-microscopy as the non-septal (old) pole. Deletions in Msesx1 prevented polar localization of tagged proteins, indicating the need for specific protein-protein interactions in polar trafficking. Remarkably, expression of the Mtbesx1 locus in Msesx1 mutants restored polar localization of tagged proteins, indicating establishment of the MtbESX-1 apparatus in M. smegmatis. This observation illustrates the cross-species conservation of protein interactions governing assembly of ESX-1, as well as polar localization. Importantly, we describe novel non-esx1-encoded proteins, which affect ESX-1 activity, which colocalize with ESX-1, and which are required for ESX-1 recruitment and assembly. This analysis provides new insights into the molecular assembly of this important determinant of Mtb virulence.

Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility.

Cells use both deterministic and stochastic mechanisms to generate cell-to-cell heterogeneity, which enables the population to better withstand environmental stress. Here we show that, within a clonal population of mycobacteria, there is deterministic heterogeneity in elongation rate that arises because mycobacteria grow in an unusual, unipolar fashion. Division of the asymmetrically growing mother cell gives rise to daughter cells that differ in elongation rate and size. Because the mycobacterial cell division cycle is governed by time, not cell size, rapidly elongating cells do not divide more frequently than slowly elongating cells. The physiologically distinct subpopulations of cells that arise through asymmetric growth and division are differentially susceptible to clinically important classes of antibiotics.

Specific pathogen detection using bioorthogonal chemistry and diagnostic magnetic resonance.

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.

Nanoporous elements in microfluidics for multiscale manipulation of bioparticles.

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.

A fluorophore ligase for site-specific protein labeling inside living cells.

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.

Fluorescent probes for super-resolution imaging in living cells.

In 1873, Ernst Abbe discovered that features closer than approximately 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.

Protein-protein interaction detection in vitro and in cells by proximity biotinylation.

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.

Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.

Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

Cells in fluidic environments are sensitive to flow frequency.

Virtually all cells accommodate to their mechanical environment. In particular, cells subject to flow respond to rapid changes in fluid shear stress (SS), cyclic stretch (CS), and pressure. Recent studies have focused on the effect of pulsatility on cellular behavior. Since cells of many different tissue beds are constantly exposed to fluid flows over a narrow range of frequencies, we hypothesized that an intrinsic flow frequency that is optimal for determining cell phenotype exists. We report here that cells from various tissue beds (bovine aortic endothelial cells (BAEC), rat small intestine epithelial cells (RSIEC), and rat lung epithelial cells (RLEC)) proliferate maximally when cultured in a perfusion bioreactor under pulsatile conditions at a specific frequency, independent of the applied SS. Vascular endothelial and pulmonary epithelial cell proliferation peaked under 1 Hz pulsatile flow. In contrast, proliferation of gastrointestinal cells, which in their physiological context are subject to no flow or higher wavelength signal, was maximum at 0.125 Hz or under no flow. Moreover, exposure of BAEC to pulsatile flow of varying frequency influenced their nitric oxide synthase activity and prostacyclin production, which reached maximum values at 1 Hz. Notably, the "optimal" frequencies for the cell types examined correspond to the physiologic operating range of the organs from where they were initially derived. These findings suggest that frequency, independent of shear, is an essential determinant of cell response in pulsatile environments.