Integrating MALDI-TOF Mass Spectrometry with Machine Learning Techniques for Rapid Antimicrobial Resistance Screening of Foodborne Bacterial Pathogens.

Although MALDI-TOF mass spectrometry (MS) is considered as the gold standard for rapid and cost-effective identification of microorganisms in routine laboratory practices, its capability for antimicrobial resistance (AMR) detection has received limited focus. Nevertheless, recent studies explored the predictive performance of MALDI-TOF MS for detecting AMR in clinical pathogens when machine learning techniques are applied. This chapter describes a routine MALDI-TOF MS workflow for the rapid screening of AMR in foodborne pathogens, with Campylobacter spp. as a study model.

Complex In Silico RNA Design with MoiRNAiFold.

In the advent of the RNA therapeutics and diagnostics era, it is of great relevance to introduce new and more efficient RNA technologies that prove to be effective tools in practical contexts. Moreover, it is of utmost importance to develop and provide access to computational tools capable of designing such RNA constructs. Here we introduce one such novel diagnostics technology (Apta-SMART) and show how to design (using MoiRNAiFold) and implement it, step by step. Moreover, we show how to combine this technique with well-known RNA amplification methods and briefly mention some encouraging results.

Quantitative Blood Serum IVDr NMR Spectroscopy in Clinical Metabolomics of Cancer, Neurodegeneration, and Internal Medicine.

Despite more than two decades of metabolomics having joined the "omics" scenery, to date only a few novel blood metabolite biomarkers have found their way into the clinic. This is changing now by massive large-scale population metabolic phenotyping for both healthy and disease cohorts. Here, nuclear magnetic resonance (NMR) spectroscopy is a method of choice, as typical blood serum markers can be easily quantified and by knowledge of precise reference concentrations, more and more NMR-amenable biomarkers are established, moving NMR from research to clinical application. Besides customized approaches, to date two major commercial platforms have evolved based on either 600 MHz (14.1 Tesla) or 500 MHz (11.7 Tesla) high-field NMR systems. This chapter provides an introduction into the field of quantitative in vitro diagnostics research (IVDr) NMR at 600 MHz and its application within clinical research of cancer, neurodegeneration, and internal medicine.

Application of a Computational Metabolomics Workflow for the Diagnosis of Inborn Errors of Metabolism in a Laboratory Setting.

Inborn errors of metabolism constitute a set of hereditary diseases that impose severe medical and physical challenges in the affected individual, in particular, for the pediatric patient population. Timely diagnosis is crucial for these patients, as any delay could result in irreversible health damage, underscoring the importance of early initiation of personalized treatment. Current routine diagnostic screening for inborn errors of metabolism relies on various targeted analyses of established biomarkers. However, this approach is time-consuming, focuses on a limited number of tests (based on clinical information) with a relatively small number of biomarkers, and does not facilitate the identification of new markers. In contrast, untargeted metabolomics-based screening offers a more efficient diagnostic solution, by assessing thousands of metabolites across multiple metabolic pathways in a single test. This not only saves time but also conserves resources for clinicians, the diagnostic laboratory, and for patients.This chapter describes the computational workflow of our "Next Generation Metabolic Screening" approach, which is a metabolomics-based method that is currently applied at the Translational Metabolic Laboratory of the Radboud University Medical Center (the Netherlands) for the diagnosis of inborn errors of metabolism.

Real-road NOx and CO2 emissions of city and highway China-6 heavy-duty diesel vehicles.

The emission of heavy-duty vehicles has raised great concerns worldwide. The complex working and loading conditions, which may differ a lot from PEMS tests, raised new challenges to the supervision and control of emissions, especially during real-world applications. On-board diagnostics (OBD) technology with data exchange enabled and strengthened the monitoring of emissions from a large number of heavy-duty diesel vehicles. This paper presents an analysis of the OBD data collected from more than 800 city and highway heavy-duty vehicles in China using remote OBD data terminals. Real-world NOx and CO2 emissions of China-6 heavy-duty vehicles have been examined. The results showed that city heavy-duty vehicles had higher NOx emission levels, which was mostly due to longer time of low SCR temperatures below 180°C. The application of novel methods based on 3B-MAW also found that heavy-duty diesel vehicles tended to have high NOx emissions at idle. Also, little difference had been found in work-based CO2 emissions, and this may be due to no major difference were found in occupancies of hot running.

Enhancing multiplex detection capabilities of the Cas12a/blocker DNA system.

In the field of molecular diagnostics, the demand for multiplex detection, aimed at reducing overall analysis costs and streamlining procedures, is on the rise, prompting ongoing developments in various technologies. In this study, we developed a novel system, the split T7 promoter-based three-way junction-transcription, coupled with Cas12a/Blocker DNA (T3-CaB), for the multiplex detection of target nucleic acids. The T3-CaB system builds upon the foundation of the T3 system, generating numerous RNA transcripts upon encountering target nucleic acids. Subsequently, these RNA transcripts displace the blocker DNA from reporter DNA, allowing active Cas12a to engage in efficient trans-cleavage reaction on the reporter DNA, resulting in a strong fluorescence signal. Importantly, the proposed system operates at the isothermal condition (37 °C), with the entire analysis completed within 90 min. Further, the detection performance of the proposed system surpasses that of the preceding Cas12a/Blocker DNA system. Model targets, namely the 16S rRNA of Staphylococcus aureus and Escherichia coli, were selected, and a successful demonstration of multiplex detection was achieved. This technology holds promise for broadening the applicability of CRISPR/Cas-based diagnostics, especially in settings necessitating multiplex detection capabilities.

Aetiologies of bacterial tick-borne febrile illnesses in humans in Africa: diagnostic limitations and the need for improvement.

Tick-borne febrile illnesses caused by pathogens like Anaplasma spp., Bartonella spp., Borrelia spp., Ehrlichia spp., Coxiella burnetii, Francisella tularensis, and Rickettsia spp., are significant health concerns in Africa. The epidemiological occurrence of these pathogens is closely linked to the habitats of their vectors, prevalent in rural and semi-urban areas where humans and livestock coexist. The overlapping clinical presentations, non-specific symptoms, and limited access to commercially available in vitro diagnostics in resource-limited settings exacerbate the complexity of accurate diagnoses. This review aimed to systematically extract and analyze existing literature on tick-borne febrile illnesses in Africa, highlighting the diagnostic challenges and presenting an up-to-date overview of the most relevant pathogens affecting human populations. A comprehensive literature search from January 1990 to June 2024 using databases like PubMed, Cochrane Library, Science Direct, EMBASE, and Google Scholar yielded 13,420 articles, of which 70 met the inclusion criteria. Anaplasma spp. were reported in Morocco, Egypt, and South Africa; Francisella spp. in Kenya and Ethiopia; Ehrlichia spp. in Cameroon; Bartonella spp. in Senegal, Namibia, South Africa, and Ethiopia; Borrelia spp. in Senegal, Gabon, Tanzania, and Ethiopia; Coxiella burnetii in 10 countries including Senegal, Mali, and South Africa; and Rickettsia spp. in 14 countries including Senegal, Algeria, and Uganda. Data were analyzed using a fixed-effect model in R version 4.0.1 and visualized on an African map using Tableau version 2022.2. This review highlights the urgent need for improved diagnostics to better manage and control tick-borne febrile illnesses in Africa.

Advancements in rapid diagnostics and genotyping of Piscirickettsia salmonis using Loop-mediated Isothermal Amplification.

Introduction: Piscirickettsia salmonis, the causative agent of Piscirickettsiosis, poses a significant threat to the Chilean aquaculture industry, resulting in substantial economic losses annually. The pathogen, first identified as specie in 1992, this pathogen was divided into two genogroups: LF-89 and EM-90, associated with different phenotypic mortality and pathogenicity. Traditional genotyping methods, such as multiplex PCR, are effective but limited by their cost, equipment requirements, and the need for specialized expertise. Methods: This study validates Loop-mediated Isothermal Amplification (LAMP) as a rapid and specific alternative for diagnosing P. salmonis infections. We developed the first qPCR and LAMP assay targeting the species-conserved tonB receptor gene (tonB-r, WP_016210144.1) for the specific species-level identification of P. salmonis. Additionally, we designed two genotyping LAMP assays to differentiate between the LF-89 and EM-90 genogroups, utilizing the unique coding sequences Nitronate monooxygenase (WP_144420689.1) for LF-89 and Acid phosphatase (WP_016210154.1) for EM-90. Results: The LAMP assays demonstrated sensitivity and specificity comparable to real-time PCR, with additional benefits including rapid results, lower costs, and simplified operation, making them particularly suitable for field use. Specificity was confirmed by testing against other salmonid pathogens, such as Renibacterium salmoninarum, Vibrio ordalii, Flavobacterium psychrophilum, Tenacibaculum maritimum, and Aeromonas salmonicida, with no cross-reactivity observed. Discussion: The visual detection method and precise differentiation between genogroups underscore LAMP's potential as a robust diagnostic tool for aquaculture. This advancement in the specie detection (qPCR and LAMP) and genotyping of P. salmonis represents a significant step forward in disease management within the aquaculture industry. The implementation of LAMP promises enhanced disease surveillance, early detection, and improved management strategies, ultimately benefiting the salmonid aquaculture sector.

Invited Review: Improved control of Johne's disease in dairy cattle through advancements in diagnostics, testing and management of young stock.

Johne's disease (JD; paratuberculosis) control programs have been regionally implemented across the globe, but few have successfully eradicated the pathogen (Mycobacterium avium ssp. paratuberculosis (MAP)) causing this disease. The limited success may partly be attributed to excluding young stock (calves and replacement heifers or bulls) from testing strategies aimed at identifying MAP-infected cattle. Young stock can shed MAP in feces and can have detectable MAP-specific antibodies in blood, as confirmed in experimentally and naturally infected cattle. Furthermore, MAP transmission causes new infections in young stock. Calves and heifers are often included in JD management strategies on dairy farms but excluded from conventional diagnostic tests due to a presumed lag between infection and detection of MAP shedding and/or MAP-specific serum antibodies. We summarize evidence of MAP shedding early in the course of infection and discuss promising diagnostics, testing and management strategies to support inclusion of young stock in JD control programs. Improvements in fecal Polymerase Chain Reaction, interferon-gamma release assay (IGRA), and enzyme-linked immunosorbent assay (ELISA) enable earlier detection of MAP and specific early immune responses. Studies on IGRA and ELISA have focused on evaluation of new antigens and optimal age of testing. There are new diagnostics, including phage-based tests to detect viable MAP, and gene expression patterns and metabolomics to detect MAP-infected young stock. In addition, refinements in testing and management of calves and heifers may enable reductions in MAP prevalence. We provide recommendations for dairy farmers, researchers, veterinarians, and other stakeholders that may improve JD control programs with an objective to control and potentially eradicate JD. Additionally, we have identified the most pressing gaps in knowledge that currently hamper inclusion of young stock in JD prevention and control programs. In summary, transmission among young stock may cause new MAP infections, and appropriate use of new diagnostic tests, testing and management strategies for young stock may improve the efficacy of JD control programs.

Extracellular vesicles.

Cells, pathogens, and other systems release extracellular vesicles (EVs). The particles promote intercellular communication and contain proteins, lipids, RNA and DNA. Initially considered to be cellular waste in the twentieth century, EVs were becoming recognized for their function in biological communication and control. EVs are divided into many subtypes: exosomes, microvesicles, and apoptotic bodies. Exosomes form in the late endosome/multivesicular body and are released when the compartments fuse with the plasma membrane. Microvesicles are generated by direct budding of the plasma membrane, whereas apoptotic bodies are formed after cellular apoptosis. The new guideline for EVs that describes alternate nomenclature for EVs. The particles modulate the immune response by affecting both innate and adaptive immunity, and their specific the structure allows them to be used as biomarkers to diagnose a variety of diseases. EVs have a wide range of applications, for example, delivery systems for medications and genetic therapies because of their ability to convey specific cellular material. In anti-tumor therapy, EVs deliver therapeutic chemicals to tumor cells. The EVs promote transplant compatibility and reduce organ rejection. Host-parasite interactions, therapeutic and diagnostic for cancer, cardiovascular disease, cardiac tissue regeneration, and the treatment of neurological diseases such as Alzheimer's and Parkinson's. The study of EVs keeps on expanding, revealing new functions and beneficial options. EVs have the potential to change drug delivery, diagnostics, and specific therapeutics, creating a new frontier in biomedical.

Assessing infectiousness and the impact of effective treatment to guide isolation recommendations for people with pulmonary tuberculosis.

Determining the extent and duration of infectiousness of individuals with pulmonary tuberculosis (TB) is critical for various aspects of TB care, including decisions regarding isolation. Studies suggest considerable heterogeneity in infectiousness of people with pulmonary TB. Pre-treatment, measures of bacillary burden including sputum smear microscopy, culture time-to-positivity, and Xpert MTB/RIF cycle threshold (Ct) value, predict the risk of transmission to contacts. Index patients with smear negative disease pose lower infectious risk than those who have smear-positive disease, and household contact infection is more likely with index patients who have lower Xpert Ct values. Newer tools that enable detection of Mycobacterium tuberculosis complex (Mtb complex) from cough aerosol sampling and face mask sampling may be better predictors of contact infection risk. Clinical factors such as cough strength and frequency, and presence of cavitation on chest imaging, may also assist with risk prediction. Post-treatment, smear and culture status are poor predictors of infectiousness. While the exact duration of infectiousness post treatment initiation remains uncertain, data from human-to-guinea pig transmission studies and clinical studies suggest effective treatment results in a rapid decline in infectiousness, irrespective of smear or culture conversion. This is largely supported by early bactericidal activity and transcriptomic studies, and cough aerosol sampling studies, although a subset of patients may have persistent cough aerosol positivity. These findings can enable a more nuanced approach to isolation decision making, while further research studies are awaited.

Blood-Based Biomarkers in Alzheimer's Disease: Advancing Non-Invasive Diagnostics and Prognostics.

Alzheimer's disease (AD), the most prevalent form of dementia, is expected to rise dramatically in incidence due to the global population aging. Traditional diagnostic approaches, such as cerebrospinal fluid analysis and positron emission tomography, are expensive and invasive, limiting their routine clinical use. Recent advances in blood-based biomarkers, including amyloid-beta, phosphorylated tau, and neurofilament light, offer promising non-invasive alternatives for early AD detection and disease monitoring. This review synthesizes current research on these blood-based biomarkers, highlighting their potential to track AD pathology and enhance diagnostic accuracy. Furthermore, this review uniquely integrates recent findings on protein-protein interaction networks and microRNA pathways, exploring novel combinations of proteomic, genomic, and epigenomic biomarkers that provide new insights into AD's molecular mechanisms. Additionally, we discuss the integration of these biomarkers with advanced neuroimaging techniques, emphasizing their potential to revolutionize AD diagnostics. Although large-scale validation is still needed, these biomarkers represent a critical advancement toward more accessible, cost-effective, and early diagnostic tools for AD.

Fusion of Raman and FTIR Spectroscopy Data Uncovers Physiological Changes Associated with Lung Cancer.

Due to the high mortality rate, more effective non-invasive diagnostic methods are still needed for lung cancer, the most common cause of cancer-related death worldwide. In this study, the integration of Raman and Fourier-transform infrared spectroscopy with advanced data-fusion techniques is investigated to improve the detection of lung cancer from human blood plasma samples. A high statistical significance was found for important protein-related oscillations, which are crucial for differentiating between lung cancer patients and healthy controls. The use of low-level data fusion and feature selection significantly improved model accuracy and emphasizes the importance of structural protein changes in cancer detection. Although other biomolecules such as carbohydrates and nucleic acids also contributed, proteins proved to be the decisive markers found using this technique. This research highlights the power of these combined spectroscopic methods to develop a non-invasive diagnostic tool for discriminating lung cancer from healthy state, with the potential to extend such studies to a variety of other diseases.

The Gut Microbiome Advances Precision Medicine and Diagnostics for Inflammatory Bowel Diseases.

The gut microbiome emerges as an integral component of precision medicine because of its signature variability among individuals and its plasticity, which enables personalized therapeutic interventions, especially when integrated with other multiomics data. This promise is further fueled by advances in next-generation sequencing and metabolomics, which allow in-depth high-precision profiling of microbiome communities, their genetic contents, and secreted chemistry. This knowledge has advanced our understanding of our microbial partners, their interaction with cellular targets, and their implication in human conditions such as inflammatory bowel disease (IBD). This explosion of microbiome data inspired the development of next-generation therapeutics for treating IBD that depend on manipulating the gut microbiome by diet modulation or using live products as therapeutics. The current landscape of artificial microbiome therapeutics is not limited to probiotics and fecal transplants but has expanded to include community consortia, engineered probiotics, and defined metabolites, bypassing several limitations that hindered rapid progress in this field such as safety and regulatory issues. More integrated research will reveal new therapeutic targets such as enzymes or receptors mediating interactions between microbiota-secreted molecules that drive or modulate diseases. With the shift toward precision medicine and the enhanced integration of host genetics and polymorphism in treatment regimes, the following key questions emerge: How can we effectively implement microbiomics to further personalize the treatment of diseases like IBD, leveraging proven and validated microbiome links? Can we modulate the microbiome to manage IBD by altering the host immune response? In this review, we discuss recent advances in understanding the mechanism underpinning the role of gut microbes in driving or preventing IBD. We highlight developed targeted approaches to reverse dysbiosis through precision editing of the microbiome. We analyze limitations and opportunities while defining the specific clinical niche for this innovative therapeutic modality for the treatment, prevention, and diagnosis of IBD and its potential implication in precision medicine.

[Disproportionately high loss in speech intelligibility]. / Disproportional hoher Verlust an Sprachverstehen.

BACKGROUND: The influence of hearing impairment on everyday hearing can be estimated by speech audiometry. There is a great deal of variability in the dependence of word recognition scores on pure-tone hearing loss. MATERIALS AND METHODS: A large clinical database of 28,261 records with complete tone and speech audiometry data was analyzed. The maximum monosyllabic word recognition score was evaluated as a function of hearing loss. Its distribution was analyzed in detail. RESULTS: In a rank analysis, the distribution of percentiles was determined as a function of pure-tone hearing loss up to 80 dBHL. CONCLUSION: The percentiles of the distribution of maximum word recognition scores for a given pure-tone hearing loss derived here can be used as reference values for a disproportionately high loss of speech recognition.

Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

BACKGROUND: We observed a discrepancy between dengue NS1 antigen test and molecular diagnostics, with the emergence of (DENV) serotype 3 in Sri Lanka and sought to understand the cause for the rise in cases and high failure rates of molecular diagnostics. METHODS: Whole genomic sequencing was carried out in 22 DENV-3 samples. Phylogenetic and molecular clock analysis were done for genotype assignment and to understand the rate of evolution. Mutation analysis was done to understand the reasons for PCR non-detection. RESULTS: We identified two DENV-3 genotypes (I and III) co-circulating. DENV-3 genotype III strains shared a common ancestor with a sequence from India collected in 2022, while DENV-3 genotype I, was found to share a common ancestor with DENV-3 sequences from China. DENV-3 genotype III was detected by the modified CDC DENV-3 primers whereas, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. We identified point mutations, C744T and A756G of the forward primer binding sites and in position G795A of the reverse primer binding sites which were not identified in DENV-3 genotype III. Furthermore, our Sri Lankan DENV-3 strains demonstrated a high root to tip ratio, compared to the previous DENV-3 sequences, indicating a high mutation rate during the points of sampling (year 2017 to 2023). CONCLUSION: The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Diagnostic Effects of Omitting Systematic Biopsies in Prostate Cancer Screening.

BACKGROUND AND OBJECTIVE: The optimal biopsy strategy in prostate cancer screening is unknown. This study aims to assess the diagnostic effects of omitting systematic biopsies in a screening cohort. METHODS: We used data from the STHLM3-MRI trial. A total of 7609 men aged 50-74 yr were randomised to undergo magnetic resonance imaging (MRI) if having an elevated risk of prostate cancer (prostate-specific antigen [PSA] ≥3 ng/ml or Stockholm3 ≥11%). Participants with Prostate Imaging Reporting and Data System (PI-RADS) ≥3 underwent targeted and systematic biopsies. Cancer detection rates from combined and targeted-only biopsies were presented as a risk ratio (RR). Subgroup analyses were stratified by age, PSA density (PSAd), and PI-RADS. Differences in reclassification rates at radical prostatectomy were calculated. KEY FINDINGS AND LIMITATIONS: The median age of the participants was 66 yr (interquartile range: 61-71) and PSA 3.8 ng/ml (2.9-5.8). Out of 395 men undergoing combined biopsies, 52 (13.2%) had International Society of Urological Pathology (ISUP) grade group (GG) 1 and 230 (58%) had ISUP GG ≥2 prostate cancer. Omission of systematic biopsies reduced cancer detection rates (RR of ISUP GG 1: 0.83 [95% confidence interval 0.64-1.07]; ISUP GG ≥2: 0.85 [0.81-0.90]; and ISUP GG ≥3: 0.86 [0.79-0.95]). Each case of averted ISUP GG 1 cancer was associated with 3.8 cases of missed ISUP GG ≥2 and 1.1 case of ISUP GG ≥3 cancer. Detection of fewer ISUP GG ≥2 cases than the number of avoided ISUP 1 cancer cases was observed in all subgroups when systematic biopsies were omitted. Using PSAd ≥0.05 ng/ml2 as a cut-off for a biopsy resulted in the same numbers of ISUP GG 1 tumours saved, with higher detection rates of ISUP GG ≥2 tumours. In 146 men undergoing radical prostatectomy, 46 (31.5%) versus 28 (19.2%) were upgraded following targeted biopsies versus a combined biopsy strategy (p < 0.05). CONCLUSIONS AND CLINICAL IMPLICATIONS: Complete omission of systematic biopsies in prostate cancer screening is associated with decreased detection of significant cancer, while reducing overdetection of insignificant cancer to a smaller extent. This strategy also increased the risk of histopathological misclassification. PATIENT SUMMARY: In a prostate cancer screening setting, we examined the diagnostic effects of systematic biopsies in addition to targeted biopsies in men with suspicious magnetic resonance imaging lesions. We found that exclusion of systematic biopsies led to reduced detection of clinically significant prostate cancer. Our findings emphasise the importance of incorporating systematic biopsies alongside targeted biopsies for improved diagnostic outcomes.

Smartphone-based thermal imaging for pedicled skin flaps: a pilot study toward objective perfusion assessment in facial, head, and neck reconstruction.

PURPOSE: This study aims to develop a practical algorithm for utilizing smartphone-based thermal imaging (SBTI) in the perioperative setting and to establish a standardized evaluation method for objectively assessing SBTI images for cutaneous perfusion of pedicled flaps in the face, head, and neck. METHODS: This prospective conducted study, integrated SBTI into the assessment of 16 patients undergoing reconstructive surgery for face and neck defects. Thermal images were captured at four timepoints: after marking (T1), after flap elevation (T2), upon completion of surgery (T3), and 24 h postoperatively (T4). The flap areas were divided into three flap zones and graded based on temperature differences (ΔT), with a grading system where grade 1 indicated perfect perfusion and grades 2 to 5 indicated increasing perfusion impairment. RESULTS: 6 male and 10 female patients aged 64-93 years (mean 78.5 years) undergoing reconstructive pedicled skin flap surgery for facial and neck defects (1 × 1 cm to 11 × 8 cm) due to diagnoses of malignant cutaneous lesions were investigated. Intraoperative assessments indicated good perfusion across flaps. One postoperative dehiscence occurred in an 83-year-old male with a cervical advancement flap, correlated with a significant temperature difference (ΔT > 4 °C) intraoperatively. Statistical analysis revealed a strong positive correlation (p = 0.0003) between clinical assessment grades and ΔT values between specific flap zones at T3. CONCLUSION: SBTI is an easy-to-apply, low-cost, real-time and reproducible technique for indirect perfusion assessment in pedicled skin flaps of the head and neck region. Further studies are needed implementing this methodology in large and free flaps.

Direct and Ultrasensitive Bioluminescent Detection of Intact Respiratory Viruses.

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of the intact virus. Instead, specific detection of intact respiratory virus particles may be more effective at assessing the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of "free" viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1-0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments, and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per µL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.