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PubMed2026

'Truthsets' for clinical validation of large-scale functional assays: Practice recommendations from Cancer Variant Interpretation Group UK (CanVIG-UK).

BACKGROUND: Large-scale functional assays, including multiplex assays of variant effect, have substantial potential to resolve variants of uncertain significance (VUS), particularly for rare missense variants where clinical and population evidence are limited. The ClinGen assay-level clinical validation framework described by Brnich et al provided baseline guidance for the use of functional data for variant classification. However, clear consensus regarding construction of variant 'truthsets' by which to clinically validate functional data remains lacking. METHODS: CanVIG-UK developed consensus recommendations for truthset construction through an iterative national consultation process involving the CanVIG Steering Advisory Group (CStAG), wider CanVIG-UK membership, and engagement with international functional genomics experts. Consultation was based on previous analyses of 2,120 truthset constructions examining the impact of truthset composition on evidence point allocation within the ClinGen assay-level clinical validation framework. RESULTS: Across several consultations, CanVIG-UK established nine guiding principles and seven best-practice recommendations for assay-level clinical validation, using the assumed context of an assay for a cancer susceptibility gene where loss-of-function is the mechanism of pathogenicity. The principal recommendation stipulates, where assays are intended for use in interpretation of largely missense variants, the truthset used to validate should comprise only missense variants. Rather than mixtures of different variant types which may serve to over-estimate assay performance. Additional recommendations support option for relaxation of truthset stringency to improve power, augmentation of benign missense truthsets with systematically derived 'proxy-clinical' benign variants, independent clinical validation separate from assayist-defined validation, and careful evaluation of missense score distributions against that of protein-truncating and synonymous variants. Guidance is also provided for scenarios with limited pathogenic truthset availability and for assays reporting multiple deleterious zones or readouts. CONCLUSIONS: The CanVIG-UK principles and recommendations for truthset construction upon the ClinGen assay-level clinical validation framework, while aiming to form a baseline for future discussion regarding other functional and disease contexts and helping to address the gap between publication of new data and routine clinical implementation.

Journal Article↗

PubMed2025

Multiplex PCR assay for the rapid detection of Klebsiella pneumoniae pathotypes.

Introduction. Klebsiella pneumoniae (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence.Gap Statement. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision.Aim. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance.Methodology. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (rmpA, rmpA2, iucA, peg344 and iroB), carbapenem-resistant (bla NDM, bla OXA-48-like and bla KPC) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity.Results. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl-1. Additionally, validation through sequencing confirmed the accuracy of detected amplicons.Conclusion. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.

Klebsiella pneumoniae↗

PubMed2025

Creating an atlas of variant effects to resolve variants of uncertain significance and guide cardiovascular medicine.

Cardiovascular diseases are leading global causes of death and disability, often presenting as interrelated phenotypes of atherosclerotic vascular disease, heart failure and arrhythmias. Cardiovascular diseases arise from interactions between environmental factors and predisposing genotypes and include common Mendelian lipid disorders, cardiomyopathies and arrhythmia syndromes. The identification of a pathogenic variant through genetic testing can inform disease diagnosis, risk prediction, treatment and family screening. However, a major roadblock in genomic medicine is that for many variants, especially missense variants, we lack sufficient evidence to enable a definitive classification, and therefore these variants are deemed as 'variants of uncertain significance'. In this Review, we describe how multiplexed assays of variant effects can enable the functional assessment of nearly all coding variants in a target sequence, potentially offering a proactive approach to identifying the functional significance of gene variants that are observed later in a patient. We discuss validation, including the role of in silico variant effect predictors, and how multiplexed experimental methods are informing cardiovascular disease biology and ultimately resolving the problem of variants of uncertain significance at scale.

Humans↗

PubMed2025

Multiplexed Dual-Color Fluorescence-Based Distinction Between Nuclear Trapping and Translocation of FOXO3.

FOXO3 is a transcription factor that mainly exerts its functions in the cell nucleus. The amino acid sequence of FOXO3 contains a nuclear localization sequence (NLS) and a nuclear export sequence (NES) allowing for nuclear/cytoplasmic shuttling that plays an important role in regulating FOXO3 activity. Nuclear accumulation of FOXO3 proteins can be the result of translocation to the nucleus triggered by upstream regulatory input or trapping of FOXO3 within the nucleus through the inhibition of its nuclear export via the receptor CRM1. In order to distinguish these two modes of FOXO3 activation, we have generated a multiplexed assay. The development of this platform includes a reporter cell line that monitors CRM1 activity by using RFP-labeled HIV-1 Rev. protein with a strong heterologous NES. Simultaneously, the intracellular localization of FOXO3 can be monitored by a second cell line stably expressing GFP-FOXO3. Here we describe a detailed protocol on how to co-culture these reporter cell lines and use them to interrogate compound-induced FOXO3 activation in order to understand the mode of action.

Forkhead Box Protein O3↗

PubMed2026

Systemic biomarkers of treatment response to methotrexate in people with painful knee osteoarthritis: A biological substudy of the PROMOTE randomised controlled clinical trial.

OBJECTIVE: Stratification of therapeutic responses may help identify efficacious therapies for osteoarthritis (OA). In the PROMOTE randomised trial, participants with elevated baseline high-sensitivity C-reactive protein (hs-CRP) showed greater pain reduction after methotrexate treatment. We set out to interrogate a broader panel of serum/plasma inflammatory response markers relevant to methotrexate actions as potential biomarkers of therapeutic effect. Our objectives were to: (i) characterize changes in these systemic markers during methotrexate treatment; determine whether (ii) baseline levels or (iii) changes in any marker during treatment were associated with treatment response; and (iv) compare these findings with the more established clinical inflammatory marker, hs-CRP. DESIGN: Plasma/serum samples from participants in PROMOTE's biological substudy were analysed for 35 inflammatory markers at baseline (pre-treatment) and at 6-months (post-treatment), by MesoScale V-plex multiplex assay. Those with paired biological and clinical data at both baseline and 6-months were included in the substudy analysis set. Relationships between markers and overall data structure were assessed by Pearson correlation and Principal Component analysis. Associations between markers (baseline levels or change over time) and change in average knee pain severity in past week (numerical rating scale, NRS) were evaluated by univariable linear regression, adjusting for baseline age, sex, and body mass index. Least Absolute Shrinkage and Selection Operator (LASSO) regression with bootstrap resampling enabled marker selection. Benjamini-Hochberg correction adjusted for multiple testing (Padj). RESULTS: 87 participants with paired blood marker and clinical data were eligible for substudy analysis. 18/35 markers were quantifiable and analysed. Systemic IL-8 and TNF-α levels decreased (Padj=0.015, 0.048 respectively) while IL-15 increased (Padj=0.033) with methotrexate treatment over 6-months. Analysing within this active treatment randomised arm, higher baseline IFN-γ was associated with greater reduction in NRS pain change (0.66 [0.01, 1.31], P=0.047), as was decreasing TNF-α over 6-months (2.25 [0.00, 4.5], P=0.049). LASSO identified higher IFN-γ, lower plasma IL-15 and IL-16, and younger age as the most important baseline predictors of pain improvement. hs-CRP was highly selected by LASSO for treatment response in both arms. In a secondary univariate treatment arm-by-biomarker interaction analysis, of the 19 markers, only hs-CRP showed consistent effects in adjusted models (at baseline, coeffic. 2.34 [0.53, 4.15], P=0.001; change over 6-months, (0.36 [0.06, 0.66], P=0.018). CONCLUSIONS: Blood measurement of IFN-γ, TNF-α, IL-15 and IL-16 as well as hs-CRP could act as potential markers to stratify the treatment response by average knee pain to methotrexate in knee osteoarthritis.

Humans↗

PubMed2026

Th2 bias and T-cell exhaustion characterize the immunopathology of non-tuberculous mycobacterial pulmonary disease.

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is an escalating global health concern with poorly defined immunological mechanisms, necessitating comprehensive profiling to guide therapeutic advances. We analyzed peripheral blood from 28 treatment-naïve NTM-PD patients (19 Mycobacterium avium complex, 9 Mycobacterium abscessus) and 27 matched controls using 42-marker mass cytometry (CyTOF) and Luminex multiplex assays. A random forest model identified predictive markers, while an in vitro murine macrophage model evaluated chemokine production. NTM-PD patients displayed significant immune shifts, including increased classical monocytes (CD14+ CD16-), reduced NKT-like cells (CD3+ CD56+), and elevated T-cell exhaustion markers (PD-1, TOX). This coincided with a Th1/Th2 balance shift characterized by heightened IL-13. Elevated IFN-γ-inducible chemokines CXCL9 and CXCL10 coexisted with this Th2-biased signature, indicating a complex, dysregulated inflammatory state. A model integrating immune-cell frequencies and cytokine profiles achieved robust diagnostic accuracy (AUC = 0.922) with prognostic potential. In vitro, NTM-infected macrophages produced substantial CXCL9 and CXCL10 levels relative to the LPS maximal activation benchmark, identifying them as a major cellular source. These findings propose an immunological framework wherein T-cell exhaustion and a Th2-biased microenvironment strongly correlate with NTM-PD pathogenesis. CXCL9, CXCL10, and IL-13 emerge as candidate therapeutic targets, while our predictive model offers a foundational approach for risk stratification.

Humans↗

PubMed2026

Inhaled vitamin A mitigates hyperoxia-induced acute and chronic lung damage in a neonatal rat model of bronchopulmonary dysplasia.

RATIONALE: Prevention of bronchopulmonary dysplasia (BPD) remains a critical unmet need. OBJECTIVES: We continued evaluating aerosolized vitamin A in a neonatal rat hyperoxia-injury model of BPD, testing whether lung-targeted inhalation yields superior outcomes compared to enteral or intramuscular (IM) delivery. METHODS: Rat pups were exposed to continuous 95% hyperoxia for seven days to induce lung damage. Vitamin A was administered from post-natal day (PD) 1-7 by inhalation, enteral or IM dosing. Persisting effectiveness of inhaled vitamin A was examined at PD 21, dwelling in normoxia after all interventions were discontinued at PD 7. MEASUREMENTS AND MAIN RESULTS: Comprehensive analyses include alveolar morphometrics, in vivo pulmonary function testing, assessment of hepatic vitamin A level, and quantitating gene and protein expression of relevant biomarkers of lung maturation, inflammation and damage/repair, employing singlex and multiplexed assays and whole-genome gene expression. CONCLUSIONS: Inhaled vitamin A suppressed hyperoxia-induced lung damage, with key hallmarks of BPD, including lung morphometrics and biomarkers associated with lung damage and inflammation, being effectively indistinguishable from healthy controls at both PD 7 and 21, Natural reparative processes were enhanced, with normal alveolarization and pulmonary function at PD 21. Despite raising hepatic vitamin A levels, enteral dosing was ineffective in restoring alveolar morphology at PD 7. Consistent with previous reporting, IM dosing yielded modest effect at PD 7 but with little evidence of benefit to pulmonary function at PD 21. Observation of dose-dependent effects with aerosolized vitamin A strengthens the evidence of the benefits of lung-targeted delivery, supporting further development of inhaled vitamin A as a BPD preventive strategy.

Journal Article↗

PubMed2025

acmgscaler: an R package and Colab for standardized gene-level variant effect score calibration within the ACMG/AMP framework.

MOTIVATION: A genome-wide variant effect calibration method was recently developed under the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP), following ClinGen recommendations for variant classification. While genome-wide approaches offer clinical utility, emerging evidence highlights the need for gene- and context-specific calibration to improve accuracy. Building on previous work, we have developed an algorithm tailored to converting functional scores from both multiplexed assays of variant effects (MAVEs) and computational variant effect predictors (VEPs) into ACMG/AMP evidence strengths. RESULTS: Our method is designed to deliver consistent performance across different genes and score distributions, with all variables adaptively determined from the input data, preventing selective adjustments or overfitting that could inflate evidence strengths beyond empirical support. To facilitate adoption, we introduce acmgscaler, a lightweight R package and a plug-and-play Google Colab notebook for the calibration of custom datasets. This algorithmic framework bridges the gap between MAVEs/VEPs and clinically actionable variant classification. AVAILABILITY AND IMPLEMENTATION: The R package and Colab notebook are available at https://github.com/badonyi/acmgscaler.

Software↗

PubMed2025

Inference and visualization of complex genotype-phenotype maps with gpmap-tools.

Understanding how biological sequences give rise to observable traits, that is, how genotype maps to phenotype, is a central goal in biology. Yet our knowledge of genotype-phenotype maps in natural systems is limited due to the high dimensionality of sequence space and the context-dependent effects of mutations. The emergence of Multiplex assays of variant effect (MAVEs), along with large collections of natural sequences, offer new opportunities to empirically characterize these maps at an unprecedented scale. However, tools for statistical and exploratory analysis of these high-dimensional data are still needed. To address this gap, we developed gpmap-tools (https://github.com/cmarti/gpmap-tools), a python library that integrates a series of models for inference, phenotypic imputation, and error estimation from MAVE data or collections of natural sequences in the presence of genetic interactions of every possible order. gpmap-tools also provides methods for summarizing patterns of epistasis and visualization of genotype-phenotype maps containing up to millions of genotypes. To demonstrate its utility, we used gpmap-tools to infer genotype-phenotype maps containing 262,144 variants of the Shine-Dalgarno sequence from both genomic 5'UTR sequences and experimental MAVE data. Visualization of the inferred landscapes consistently revealed high-fitness ridges that link core motifs at different distances from the start codon. In summary, gpmap-tools provides a flexible, interpretable framework for studying complex genotype-phenotype maps, opening new avenues for understanding the architecture of genetic interactions and their evolutionary consequences.

Gaussian process↗

PubMed2026

Obese adipocytes induce fibroblast-to-myofibroblast transition through TGF-β1 signaling: implications in asthma pathogenesis.

Obesity, a key risk factor for severe asthma, is associated with worsening symptoms and poor responses to conventional therapies. Recent studies have highlighted the presence of adipocytes within airway walls, which correlates positively with body mass index (BMI). However, the role of adipocytes in asthma pathogenesis remains largely unknown. This study aims to explore their potential contribution to airway fibrosis, a progressive form of the disease, through fibroblast-to-myofibroblast transition (FMT). In vitro coculture models were developed to investigate the interactions between adipocytes (derived from patients with and without obesity) and fibroblasts (from patients with and without asthma) on FMT. Proteomic and multiplex analyses were used to identify potential mediators of adipocyte-induced FMT. Our data revealed a significant increase in fibrogenic markers, such as alpha-smooth muscle actin and vimentin, in fibroblasts cocultured with obese (Ob) adipocytes. Notably, this transition was more pronounced in asthmatic fibroblasts compared with healthy fibroblasts. Proteomic profiling of cocultured Ob-adipocytes and asthmatic fibroblasts identified several significantly upregulated proteins linked to the regulation of the transforming growth factor-beta (TGF-β) signaling pathway, including inhibin A, latent TGF-β binding protein 1, thrombospondin 1, and follistatin. The role of TGF-β was further substantiated by multiplex assays, which demonstrated a significant increase in TGF-β and leptin production by Ob-adipocytes following coculture. These findings suggest that Ob-adipocytes may promote FMT in fibroblasts, especially asthmatic fibroblasts, by activating the TGF-β signaling pathway. This highlights a potential mechanism by which obesity exacerbates asthma severity and fibrosis, providing new avenues for therapeutic intervention.NEW & NOTEWORTHY Adipocytes have been found in the airway wall of patients with obesity. This study is the first to show that adipocytes derived from patients with obesity can induce features of airway remodeling that is seen in patients with asthma such as fibroblast-to-myofibroblast transition via the TGF-beta signaling pathway in an indirect mode of cellular communication. This highlights a potential mechanism by which obesity exacerbates asthma severity and fibrosis, providing new avenues for therapeutic intervention.

Humans↗

PubMed2026

Effects of exercise on muscle strength and characteristics in rheumatic diseases and sarcopenia: Protocol for the Care for Muscle (C4M) Study.

OBJECTIVE: Muscle weakness is prevalent in rheumatoid arthritis (RA), osteoarthritis (OA) and sarcopenia (SARC). Endurance exercises may improve mitochondrial function and oxidative capacity, while strength exercises are thought to stimulate myofibrillar protein synthesis. This study aims to compare the effects of strength and endurance exercise and explore the association between muscle characteristics and exercise outcomes in patients with RA, OA and SARC. We hypothesize that responses to endurance and strength exercises in patients with muscle weakness are influenced by intramuscular pathology including muscle morphology, mitochondrial function, and systemic inflammation, based on their disease pathology, potentially requiring personalized training schedules. METHODS: This two-arm, parallel-group exploratory trial will enroll 69 patients (23 RA, 23 OA, 23 SARC), randomized to endurance (n = 35) or muscle strength exercises (n = 34), using minimization to balance disease type and gender. The 8-week intervention includes two supervised sessions per week using a controlled cable pulley device (Reforter™) and fitness equipment, plus one weekly home-based session. The primary outcome is isokinetic muscle strength (peak torque), measured with the Biodex system®. Secondary outcomes include muscle morphology, mitochondrial function, systemic inflammation and muscle endurance (by Biodex and 6 Minute Walk test). Muscle morphology will be assessed via 3D ultrasound imaging of the vastus lateralis. Mitochondrial function will be analyzed using high-resolution respirometry on muscle biopsies. Systemic inflammation will be measured using multiplex assays or ELISA on serum samples. DISCUSSION: This study will explore differential responses to muscle endurance and muscle strength exercises in patients with RA, OA, and SARC, offering novel insights into the molecular mechanisms of muscle weakness. The findings may help identify potential mechanisms underlying variability in exercise response and provide effect size estimates to guide future confirmatory studies and more targeted exercise interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT06480643 (date of registration28-06-24).

Humans↗

PubMed2026

MASLD Exacerbates Chronic Low-dose PM2.5-induced Lung Injury, Inflammation, and Fibrosis.

BACKGROUND/AIM: Fine particulate matter (PM2.5) and metabolic dysfunction-associated steatotic liver disease (MASLD) are independent risk factors for respiratory disease. However, the combined impact of chronic, low-dose PM2.5 exposure and Western diet (WD)-induced metabolic dysfunction on pulmonary health remains poorly understood. We investigated whether this metabolic state exacerbates PM2.5-driven pathologies using an environmentally relevant PM2.5 dosage (~50 μg/m3). MATERIALS AND METHODS: C57BL/6J mice were fed a WD or normal diet (ND) for 28 weeks and concurrently received intratracheal instillations of PM2.5 (0.5 mg/kg diesel particulate matter) or vehicle three times per week. The MASLD phenotype was confirmed through metabolic and histological analyses. Pulmonary injury, fibrosis, and inflammation were assessed via histology (hematoxylin and eosin, and Masson's trichrome staining) and cytokine quantification in both bronchoalveolar lavage fluid using Luminex multiplex assay and lung tissue using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: The WD successfully induced MASLD characterized by weight gain, hepatic steatosis, and dyslipidemia. While PM2.5 exposure did not significantly worsen the primary features of MASLD, its combination with a WD markedly exacerbated pulmonary injury and fibrosis compared to PM2.5 exposure alone. This exacerbation was driven by a surge in pro-inflammatory chemokines, including C-X-C motif chemokine ligands 1 and 2 (CXCL1 and CXCL2), and C-C motif chemokine ligand 5 (CCL5), confirmed by Luminex analysis of lavage fluid and mRNA/protein quantification in lung tissue. CONCLUSION: Diet-induced metabolic dysfunction primes the lung for a hyper-inflammatory response to chronic PM2.5 exposure. These findings identify individuals with MASLD as a population with heightened susceptibility to air pollution-related respiratory diseases and underscore the critical interplay between metabolic health and environmental toxicology.

Animals↗

PubMed2026

Single-cell transcriptomics reveals distinct microglial state remodeling associated with the (R)-nicotine/diosmetin combination and galantamine in LPS-challenged BV2 cells.

BACKGROUND: Microglial neuroinflammation contributes to the progression of neurodegenerative diseases, yet it remains challenging to attenuate inflammatory responses while preserving cellular function. The effects of (R)-nicotine, diosmetin, their combined administration, and galantamine on heterogeneous BV2 transcriptional states have not been compared at single-cell resolution. METHODS: LPS-stimulated BV2 microglial-like cells were treated with (R)-nicotine, diosmetin, their combination (DR), or galantamine. Single-cell RNA sequencing was performed with three biological replicates per group and integrated with RNA velocity and SCENIC regulon inference to characterize treatment-associated state redistribution, inferred local transcriptional directionality and regulon-activity patterns. Functional validation included CCK-8 metabolic activity assays, multiplex cytokine ELISA, BDNF/GDNF quantification, qPCR, and high-content immunofluorescence analysis of iNOS and Arg1 at single-cell resolution. RESULTS: LPS decreased the relative abundance of the Itgae+/Plk4+ cluster while increasing the Nmur1+/Limk2+ cluster and inflammatory effector programs. DR treatment suppressed pro-inflammatory cytokine release without significantly reducing CCK-8-assessed metabolic activity, increased the Itgae+/Plk4+ cluster proportion, and increased BDNF/GDNF relative to LPS. RNA velocity and SCENIC analyses suggested that DR and galantamine showed distinct transcriptional and regulatory patterns: DR attenuated Batf-associated inflammatory regulons and was associated with increased Atf3-linked stress-response activity, whereas galantamine preferentially engaged DNA repair and genome-maintenance programs. CONCLUSION: These findings indicate that the DR condition was associated with remodeling of LPS-challenged BV2 microglial-like states, attenuation of inflammatory programs, and increased neurotrophic outputs relative to LPS, without significantly reducing CCK-8-assessed metabolic activity. This study provides a single-cell characterization of distinct treatment-associated responses to (R)-nicotine, diosmetin, their combined administration, and galantamine.

Microglia↗

PubMed2026

Clinical Validation of a Multiplex Urine Biomarker Assay for Surveillance of Recurrent Bladder Cancer.

PURPOSE: More than 50% of patients with non-muscle-invasive bladder cancer experience recurrence, requiring lifelong surveillance with repeated cystoscopy. Given the invasive nature and cost of cystoscopy, accurate noninvasive tools are needed to support risk-adapted monitoring. We evaluated the ability of Oncuria-Monitor, a multiplex urine biomarker assay, to detect recurrent bladder cancer during surveillance. PATIENTS AND METHODS: Between February 2017 and August 2020, six medical centers in the United States and Japan prospectively enrolled 300 patients with a history of bladder cancer, generating 1,248 serial urine samples. Participants were divided into training and validation cohorts. At each surveillance visit over 2 years, urine samples were analyzed in a blinded manner using Oncuria-Monitor and BladderChek, alongside urine cytology. Test performance was compared with cystoscopy and histopathology-confirmed recurrence. RESULTS: Recurrent bladder cancer was identified in 31% (93/300) of participants, with 143 total recurrences during follow-up, including 90 tumors in the validation cohort. In the validation cohort, Oncuria-Monitor achieved a sensitivity of 85.6% [95% confidence interval (CI), 78.1%-92.2%] and a negative predictive value (NPV) of 93% (95% CI, 89.2%-96.4%). In comparison, BladderChek demonstrated a sensitivity of 20.0% and an NPV of 88%, whereas urine cytology showed a sensitivity of 36.9% and an NPV of 91.6%. The number needed to evaluate to detect one recurrence was 3 for both cystoscopy and Oncuria-Monitor, compared with 15 for BladderChek and 8 for cytology. CONCLUSIONS: In this large prospective longitudinal study, Oncuria-Monitor demonstrated clinically actionable performance, enabling a rule-out strategy that could safely reduce cystoscopy in approximately 25% of surveillance visits. These findings support a paradigm shift toward biomarker-guided, risk-adapted surveillance in bladder cancer that reduces unnecessary invasive procedures while maintaining oncologic safety.

Humans↗

PubMed2026

Development of a Multiplex Polymerase Chain Reaction Assay for Differentiating Three Lactococcus Species Associated With Piscine Lactococcosis.

Piscine lactococcosis is an important bacterial disease of farmed fish. The causative agents, Lactococcus garvieae, Lactococcus petauri and Lactococcus formosensis, are closely related, which complicates species-level identification. We developed a conventional multiplex PCR assay targeting species-specific genes identified by comparative genomic analysis. Average nucleotide identity reassignment of 441 publicly available genome assemblies identified 111 L. garvieae, 255 L. petauri and 75 L. formosensis genomes. Species-specific primers and a tuf-based Lactococcus common control were evaluated using in silico polymerase chain reaction (PCR) against target genomes and 10,460 off-target assemblies representing 474 taxa in 12 genera. Experimental specificity was assessed using six target strains and 19 non-target fish pathogens. Distinct amplicons of 195, 333 and 500 bp were produced for L. garvieae, L. petauri and L. formosensis, respectively, together with a 132-bp control amplicon. No cross-amplification was observed. All target species were detected in mixed-DNA samples and spiked kidney and spleen homogenates from two fish species. Analytical detection limits were estimated based on microscopic cell counts of bacterial suspensions before DNA extraction and ranged from 0.956 to 8.55 cell equivalents per reaction. This assay represents a rapid, low-cost method for differentiating lactococcosis-causing Lactococcus species using standard PCR and agarose gel electrophoresis.

Lactococcus garvieae↗

PubMed2026

Improving quality control of microbial agri-inputs by confirming strain identity with an easy and low-cost PCR-multiplex: A study case with Azospirillum brasilense.

The first commercial product containing the Azospirillum brasilense elite strains Ab-V5 and Ab-V6 was launched in Brazil in 2009. These strains have demonstrated agronomic efficiency in grasses and in legume co-inoculation, accounting for approximately 43 million doses in 2024. Official identification of these strains is currently performed by rep-PCR, a reliable but time-consuming and laborious method. In this study, a multiplex PCR assay was developed for the simultaneous identification of Ab-V5 and Ab-V6 in a single reaction using strain-specific SNPs. Forward primers were designed so that the terminal nucleotide at the 3' end corresponded to a strain-specific SNP unique to each target strain. To further enhance specificity, artificial mismatches were introduced at the fourth nucleotide from the 3' end of the forward primers. SNPs were identified using Snippy based on genomic alignments between Ab-V5 and Ab-V6 and confirmed by local BLASTn against the genomes of other Azospirillum species. In the multiplex assay, simultaneous and specific amplification of both strains was observed in a single reaction, without non-specific amplification. Primer specificity was also experimentally evaluated against other A. brasilense strains (Ab-V1, Ab-V2, Ab-V4, Ab-V7, Ab-V8, and Sp7T), in silico against bacteria from different genera associated with agricultural inoculants, and in commercial inoculant samples containing Ab-V5 and Ab-V6. The results confirmed the high specificity of the primers for Ab-V5 and Ab-V6 and demonstrated that the assay was capable of identifying the strains in commercial inoculants. This assay facilitates inoculant quality control by enabling strain confirmation using a simple, rapid, and low-cost method.

Azospirillum↗

PubMed2026

A 29-plex MOL-PCR assay for simultaneous detection of selected major, non-typing, and accessory virulence genes in Clostridium perfringens.

Clostridium perfringens is an important pathogen of humans and animals, responsible for a broad spectrum of diseases mediated by diverse toxins and virulence factors. Precise and extended toxin-gene profiling is valuable for strain characterization and molecular epidemiological surveillance. Here, we describe the development of a 29-plex Multiple Oligonucleotide Ligation PCR (MOL-PCR) assay that enables the simultaneous detection of a large and important panel of 27 C. perfringens toxin-related genes - covering major typing toxins as well as an extended panel of non-typing and accessory virulence genes - thus moving beyond the classical toxinotyping framework. The assay was evaluated in comparison with six multiplex qPCR assays. In both systems, the gene encoding the Clostridium perfringens-specific serine O-acetyltransferase (EpsC) was used as a molecular marker for species confirmation, and an internal amplification control was included to detect potentially false-negative results. Analytical specificity testing confirmed exclusive amplification in C. perfringens and sequencing confirmed the toxin-gene profiles of reference strains. Comparative analysis of 72 reference and field isolates (1,944 data points) demonstrated complete concordance for 637 positive detections, yielding 100% positive agreement and 99.7% negative agreement relative to the comparative qPCR method. The limit of detection was 100 fg/µl (approx. 3 × 101 genome equivalents; GE) for qPCR and 1  pg/µl (approx. 3 × 102 GE) for MOL-PCR. Despite its high multiplex level, MOL-PCR showed high agreement with qPCR. The developed MOL-PCR method provides a rapid, high-throughput, and cost-effective tool for expanded toxin-gene profiling of C. perfringens isolates targeting major typing toxins and selected non-typing and accessory virulence genes. Therefore, it may support advanced toxin-gene characterization, molecular epidemiology, and One Health-oriented surveillance of evolving virulence landscapes.

Clostridium perfringens↗

PubMed2026

Multiplexed RT-LAMP Assays in Handheld Devices for In-Situ Detection of Chikungunya, Dengue, Mayaro, and Zika Viruses.

Mosquito-borne viruses pose a significant global health challenge, particularly in resource-limited settings where multiple viruses often cause illnesses with similar symptoms that require different treatment. We introduce the first 7-plex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in a hand-held device capable of detecting the presence of Chikungunya virus (CHIKV), dengue virus serotypes (DENV 1-4), Mayaro virus (MAYV), and Zika virus (ZIKV) in a single test. If the result is positive from the single-plex device for the 7-plex assay, 3-plex and 4-plex devices are then used to identify the exact virus within a specimen. In-situ detection is achieved by integrating valve-enabled, paper-based sample preparation with fluorescence detection using a blue LED flashlight as a light source and a yellow plastic film as a filter, allowing visual discrimination between positive and negative samples by the naked eye or by recording images using a smart phone. The detection limits ranged from 2 genome equivalents (GE)/reaction (for ZIKV) to 92 GE/reaction (for DENV-3) across 7 types of viruses when 1 μL of viral RNA was used. We observed 90% overall agreement between the point-of-care (POC) device and lab-based reverse transcription polymerase chain reactions (RT-PCR) when blinded clinical specimens were tested. This assay and device have a potential to address critical surveillance gaps in endemic regions, enabling timely detection of multiple mosquito-borne viruses to guide appropriate clinical management and public health countermeasures in settings where laboratory resources are scarce.

Nucleic Acid Amplification Techniques↗