Search Results (2,480)

Canine dirofilariasis, caused by Dirofilaria immitis and Dirofilaria repens, is an emerging vector-borne disease of increasing veterinary and zoonotic importance. Rapid and species-specific detection is essential for effective clinical management and epidemiological surveillance. This study aimed to develop and diagnostically evaluate two novel species-specific loop-mediated isothermal amplification (LAMP) assays for the direct detection of D. immitis and D. repens in canine whole blood, performed in parallel in separate reactions, with emphasis on simplified and potentially near-point-of-care applicability. Primers targeting mitochondrial COI and NADH gene regions were designed and validated. In silico specificity analysis against 13 filarioid species confirmed the absence of non-specific primer binding. A direct closed-tube LAMP protocol using sodium hydroxide–Chelex-100 lysis was optimized, enabling amplification without conventional DNA extraction while reducing contamination risk and processing time to under 60 min. Relative diagnostic performance was evaluated relative to quantitative real-time PCR (qPCR) results. Using purified DNA, the D. repens assay achieved 100% relative sensitivity and relative specificity, whereas the D. immitis assay showed 94.5% relative sensitivity and 100% specificity. In direct whole-blood assays, relative specificity remained 100% for both targets, while sensitivity decreased to 90.9% for D. immitis and 77.42% for D. repens, with most false-negative reactions associated with high qPCR Ct values (>30). These findings demonstrate that the proposed assays provide a rapid and practical molecular diagnostic approach with potential applicability for point-of-care veterinary testing. Full article

Escherichia coli, Clostridium perfringens and Cryptosporidium are common diarrhea-related pathogens in dairy calves, posing considerable economic losses to animal husbandry and threatening public health. A previous study in our lab found the frequent occurrence of Escherichia coli virulence genes (eaeA, stx1 and stx2), Clostridium perfringens cpa and Cryptosporidium in diarrheic dairy calves in Ningxia Hui Autonomous Region, China. The present study aimed to develop a multiplex PCR for simultaneous detection of these virulence genes and Cryptosporidium in diarrheic dairy calves. The multiplex PCR demonstrated sensitivities of 2060 copies, 18200 copies, 1300 copies, 1990 copies and 974 copies for stx1, stx2, eaeA, cpa and 18S rRNA, respectively. Moreover, the method showed no cross-reactivity with Giardia duodenalis, Enterocytozoon bieneusi, Eimeria, Haemonchus contortus, Oesophagostomum, Moniezia, Salmonella, Proteus mirabilis and Staphylococcus aureus. Further application of the multiplex PCR in 20 clinical faecal samples from diarrheic dairy calves found that the positive rates of the multiplex PCR assay were 55% (11/20), 50% (10/20), 60% (12/20), 45% (9/20) and 25% (5/20) for stx1, stx2, eaeA, cpa and 18S rRNA, respectively, which were not significantly lower than those of the conventional PCR targeting stx1 (60%, 12/20) and eaeA (65%, 13/20), but higher than those of the reported PCR targeting stx2 (45%, 9/20) and cpa (40%, 8/20), and were consistent with those of the reported nested PCR targeting 18S rRNA (25%, 5/20). Taken together, the present study preliminarily developed a multiplex PCR assay for the rapid detection of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in dairy calves, which could provide basic data and technical support for the diagnosis and prevention of calf diarrhea. However, more samples from divergent clinical settings are needed to validate the assay in the diagnosis of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in future studies. Full article

Pediatric respiratory infections remain among the leading causes of emergency department visits, hospitalization and pediatric intensive care unit (PICU) admission. Although most acute respiratory infections in children are viral, clinical manifestations overlap substantially among viral, bacterial and atypical pathogens, creating diagnostic uncertainty and promoting empirical antimicrobial use. Rapid antigen tests, nucleic acid amplification tests, multiplex respiratory panels and metagenomic sequencing have expanded the ability to detect pathogens within clinically actionable timeframes. However, evidence from pediatric emergency trials indicates that rapid pathogen detection alone does not necessarily reduce antibiotic prescribing or healthcare costs. These findings suggest that the value of rapid diagnostics depends less on analytical breadth than on whether testing is applied to the right child, in the right clinical scenario and within a predefined decision pathway. This narrative review reorganizes the evidence around a scenario-driven top-pathogen framework. Top pathogens are defined as organisms that, in a specific age group, syndrome, season or care setting, have high prevalence, severe disease potential, transmissibility, treatment implications, antimicrobial resistance relevance or infection-control value. We discuss how top-pathogen testing should differ across emergency triage, inpatient ward management, severe pneumonia, PICU care, hospital-acquired pneumonia, ventilator-associated pneumonia and outbreak settings. We further examine the economic mechanisms through which rapid testing may generate value, including reduced unnecessary antibiotics, timely antiviral therapy, optimized isolation, shorter length of stay, reduced repeated testing and prevention of healthcare-associated transmission. Finally, we propose implementation principles centered on diagnostic stewardship, antimicrobial stewardship, local epidemiology and real-world cost-effectiveness evaluation. A scenario-driven top-pathogen strategy may provide a practical bridge between broad syndromic testing and precision infectious disease management in children. Full article

Candidemia is a major healthcare-associated bloodstream infection with high mortality, requiring ongoing surveillance to guide management. This retrospective study analyzed 306 candidemia episodes diagnosed between 2015 and 2024 at a Spanish tertiary-care hospital, comparing two periods (2015–2019 vs. 2020–2024). The overall incidence was 0.79 episodes per 1.000 admissions, with peaks in 2021 and 2024. Candida albicans was the most common species (44.8%), followed by Candida parapsilosis (19.0%) and Nakaseomyces glabrata (15.7%). A significant epidemiological shift occurred in the later period, with increased C. albicans, decreased C. parapsilosis, and emergence of N. glabrata as the second-most frequent species. ICU-related cases rose significantly during the COVID-19 period. Diagnostic turnaround times improved, including faster blood culture positivity and species identification by MALDI-TOF, supported by rapid PCR testing with high sensitivity (91.7%). Antifungal resistance to fluconazole was notable in N. glabrata (48.1%). Empirical echinocandin use increased, alongside greater targeted fluconazole therapy. Antimicrobial stewardship interventions, mainly de-escalation strategies, were widely implemented after 2019. Overall mortality was 40.8%, with a decline observed in 2023–2024. These findings suggest that integrated diagnostic and stewardship strategies may improve outcomes, though causal relationships require further study. Full article

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is a major pathogen causing severe growth retardation in shrimp, leading to substantial economic losses in global aquaculture. To address the urgent need for accurate, rapid, and field-deployable diagnostic tools for EHP, this study developed a novel one-pot detection platform by integrating asymmetric Enzymatic Recombinase Amplification (aERA) with a PAM-independent CRISPR/Cas12a system (AYERA-Cas12a) based on ssDNA activation. This design circumvents the compatibility challenge between isothermal amplification and CRISPR activity in a single tube by generating single-stranded DNA amplicons that activate Cas12a without requiring a PAM sequence. The assay operates at a constant temperature of 46 °C and completes detection within 15 min. It achieves a sensitivity of 10 copies/μL, equivalent to qPCR, and shows no cross-reactivity with six other prevalent shrimp pathogens. Validation using 56 clinical shrimp (Litopenaeus vannamei, L. vannamei) samples demonstrated complete agreement with qPCR results. With its simple procedure, isothermal conditions, and clear endpoint fluorescence readout under blue light, the AYERA-Cas12a platform is suitable for point-of-care testing (POCT). This work provides a user-friendly tool for the on-site surveillance and early diagnosis of EHP, offering significant potential for improving disease management in shrimp farming. Full article

Cephalopod products are widely distributed as frozen raw materials or cut portions, making morphology-based species identification difficult during commercial handling and inspection. In this study, we developed a conventional multiplex PCR assay for the simultaneous identification of six commercially important cephalopod species, Octopus vulgaris, O. ocellatus, O. minor, Enteroctopus dofleini, Dosidicus gigas, and Todarodes pacificus. Species-specific forward primers and a shared reverse primer were designed from the mitochondrial cytochrome c oxidase subunit I (COI) region to generate distinct diagnostic amplicons within a single reaction. The assay successfully produced species-resolved bands of 459, 365, 248, 194, 141, and 82 bp for O. vulgaris, E. dofleini, O. ocellatus, O. minor, D. gigas, and T. pacificus, respectively, with no ambiguous overlap among diagnostic fragments. Clear and reproducible amplification was obtained at annealing temperatures of 51–54 °C, with 52 °C selected as the standard condition, indicating useful operational tolerance for routine application. The assay also retained consistent diagnostic performance down to 1 ng of template DNA per reaction. These results demonstrate that the developed multiplex PCR assay provides a simple, rapid, and gel-based method for the preliminary identification of selected cephalopod species in frozen commercial materials and may be useful for seafood inspection and market surveillance. Full article

Acute kidney injury (AKI) caused by ischemia–reperfusion injury (IRI) involves rapid activation of innate immune responses, in which myeloid-derived immune cells critically shape injury severity. Y-box binding protein 1 (YB-1) regulates pro-inflammatory gene expression intracellularly and can be secreted to function extracellularly, yet how these two compartments jointly influence early IRI pathology remains poorly understood. To dissect the roles of intracellular myeloid versus extracellular YB-1, we subjected myeloid-specific Ybx1 knockout, Ybx1^fl/fl × LysM^cre, mice and wild-type (WT) littermates to unilateral renal IRI following administration of either a neutralizing anti-YB-1 antibody or control IgG. Kidney injury, inflammation, immune cell recruitment, neutrophil extracellular trap (NET) formation, antibody localization, and Fcγ receptor expression were assessed by qRT-PCR, histology, immunostaining, Western blotting, and flow cytometry. Myeloid-specific knockout of Ybx1 markedly reduced renal inflammation, neutrophil infiltration, NET formation, and tubular injury. This protective phenotype was lost when extracellular YB-1 was simultaneously reduced: anti-YB-1 treatment in knockout mice restored pro-inflammatory cytokine expression, increased tubular damage markers such as NGAL and KIM-1, exacerbated neutrophil recruitment and NET formation, and led to luminar accumulation of YB-1/anti-YB-1 immune complexes in tubular cells. Mechanistically, Ybx1-deficient myeloid cells exhibited significantly reduced CD16 expression, pointing to impaired Fcγ receptor-mediated phagocytosis as the cause of defective immune complex clearance. In contrast, wild-type mice efficiently cleared extracellular YB-1 complexes and showed no injury aggravation upon antibody treatment. Our findings identify myeloid YB-1 as a central regulator of early inflammatory injury in renal IRI and reveal that its protective depletion becomes pathogenic when extracellular YB-1 is simultaneously neutralized, likely due to unmasked defects in immune complex clearance. Full article

Green mold disease caused by Trichoderma species represents one of the most serious threats to the cultivation of Ganoderma lingzhi. In this study, a rapid molecular detection system was developed for the identification of green mold caused by Trichoderma longibrachiatum. The pathogen was first identified based on morphological characteristics and pathogenicity tests. Species-specific primers were then designed targeting the RPB2 gene region, and their specificity and sensitivity were evaluated using polymerase chain reaction (PCR). The results confirmed that the causal pathogen was T. longibrachiatum. The designed primers exhibited high specificity and produced amplification only with genomic DNA from the target strain. Sensitivity assays demonstrated that the minimum detectable DNA concentration was 10⁻² ng/µL. In soil inoculation experiments, the target DNA could be detected in soil containing 4.28 × 10⁶ spores/g using the developed primers. Compared with conventional culture-based detection methods, the molecular detection system established in this study is faster, more accurate, and highly sensitive. This method provides a practical diagnostic tool for the early detection and management of green mold disease in G. lingzhi cultivation. Full article

Streptococcus agalactiae is a major contagious pathogen of bovine mastitis and causes substantial economic losses in the dairy industry. In this study, a dual-readout RPA-PfAgo detection platform targeting the conserved cfb gene of S. agalactiae was established and optimized. Seven pairs of RPA primers were designed and screened to construct the Basic-RPA assay, and six guide DNAs (gDNAs) together with a specific probe were evaluated for PfAgo-assisted detection. Field validation was performed using 153 bovine milk samples collected from five dairy-farming regions in China, and assay performance was compared with bacteriological culture and a standardized quantitative PCR (qPCR) assay. The Basic-RPA assay achieved optimal amplification at 37 °C for 30 min, with a detection limit of 1 × 10⁻³ ng/µL and no cross-reactivity with non-target bacteria. The optimized RPA-PfAgo-RTF assay detected as few as 10 copies/µL, whereas the RPA-PfAgo-LFD assay detected 100 copies/µL, and both formats showed high analytical specificity. In field milk samples, bacteriological culture detected 21 positive samples, whereas standardized real-time PCR (qPCR), RPA-PfAgo-RTF, and RPA-PfAgo-LFD each detected 33 positive samples. When compared with bacteriological culture as a conventional comparator, all three molecular assays showed 100.00% positive agreement, 90.91% negative agreement, and a Kappa value of 0.733. In addition, RPA-PfAgo-RTF and RPA-PfAgo-LFD were completely concordant with the standardized qPCR assay across all 153 samples. These results indicate that the dual-readout RPA-PfAgo platform is a rapid and reliable molecular tool for detection of S. agalactiae in bovine milk. Full article

Background: Programmed cell death protein-1 (PD-1) and its ligands, PD-L1 and PD-L2, constitute a central immune checkpoint pathway that regulates T-cell activity and tumor immune escape, while their relationship with STAT signaling remains incompletely understood in ccRCC. Methods: We analyzed 27 paired ccRCC and adjacent non-tumorous human kidney tissue samples. mRNA levels of PD-1, PD-L1, PD-L2, STAT1, and STAT3 were quantified by RT-qPCR. In addition, representative human ccRCC cell lines (CAKI-2 and A-498) were treated with IFN-γ to assess the time-dependent modulation of immune checkpoint molecules and STAT pathway activation. Results: PD-L1 and PD-L2 were significantly upregulated in tumor tissues compared with adjacent normal kidney tissue. Exploratory observation suggests grade dependent increase. Whereas PD-1 was predominantly downregulated, IFN-γ treatment induced a rapid transcriptional upregulation of PD-L1 and PD-L2 in RCC cell lines, with maximal protein accumulation observed at 72 h. STAT1, but not STAT3, exhibited dynamic induction following IFN-γ stimulation, showing temporal association with PD-L1 and PD-L2 upregulation, indicating cell-line-specific regulatory effects. Correlation analyses confirmed a strong correlation between PD-1 and its ligands, whereas STAT1 and STAT3 expression showed no direct association with PD-L1 or PD-L2 levels in cancer samples. Conclusions: Our findings demonstrate that PD-L1 and PD-L2 are frequently upregulated in ccRCC and dynamically regulated by IFN-γ/STAT1-dependent signaling. Our results provide additional insight into the mechanisms of immune escape and underscore the potential of integrated profiling of PD-1 ligands and STAT signaling to guide personalized immunotherapeutic strategies in ccRCC. Full article

Meningitis multiplex PCR (MMP) panels are increasingly used in acute bacterial meningitis (ABM), but their clinical integration remains incompletely characterized. We evaluated MMP panel implementation using a mixed-methods approach. We included 55 adults with ABM of confirmed etiology: Group 1 (MMP plus conventional microbiology, n = 25) and Group 2 (conventional methods only, n = 30). The qualitative component comprised semi-structured interviews with infectious disease specialists, analyzed using thematic analysis. Compared with Group 2, Group 1 had a significantly shorter time from lumbar puncture to diagnosis [1.9 (IQR 1.7–2.6) vs. 27.3 (18–47.2) h] and more frequent targeted therapy [19 (76%) vs. 13 (43.3%)]. However, MMP panel use was not associated with antibiotic de-escalation [11 (44%) vs. 12 (40%)], median length of hospitalization (22 days in both groups), median duration of therapy [14 (10–21) vs. 17 (11–22) days], ICU admission [11 (44%) vs. 13 (43.3%)], or mortality [2 (8%) vs. 6 (20%)]. Interviews (n = 20) identified four themes: rapid etiological clarification, perceived limitations, antimicrobial optimization and clinical integration. MMP may facilitate rapid diagnosis, but its impact on outcomes and clinical integration remains limited. Faster availability of etiological information does not necessarily improve antimicrobial decision-making in the absence of antimicrobial stewardship programs. Full article

The success of DNA amplification in Polymerase Chain Reaction (PCR) devices inherently depends on the rapid and absolute zero-overshoot temperature control of thermoelectric cooler (TEC) systems. In the literature, metaheuristic algorithms employed for proportional–integral–derivative (PID) tuning typically operate within unconstrained search spaces, relying exclusively on time-domain error metrics like ITAE. This conventional approach causes ‘gradient blindness’ and neglects frequency-domain robustness, resulting in excessive temperature overshoots that violate biological safety limits and lead to enzyme denaturation. To solve this problem, we propose a hybrid frequency-time domain optimization framework. Utilizing a first order plus dead-time (FOPDT) model for TEC dynamics, the PID search space is analytically restricted via Ziegler–Nichol’s stability boundaries. Furthermore, Phase Margin (PM ≥ 45°) and absolute zero-overshoot conditions are integrated into the objective function as a strict penalty mechanism. Evaluations conducted with five distinct metaheuristic algorithms (PSO, GWO, WOA, ABC, and ACO) prove that while traditional unconstrained methods yield overshoots up to 19.04%, the proposed architecture successfully confines all optimization agents to a globally stable region, enabling specific algorithms like ABC, PSO, and WOA to achieve exactly 0.00% overshoot. Validated across a realistic multi-step PCR cycle (95–55–75 °C), the developed robust controller settles into the denaturation phase with a 0.00 °C peak error, guaranteeing biological sample safety and delivering a reliable control framework for rapid-cycle PCR platforms. Full article

Long-read sequencing technologies provide portable and flexible service, making them attractive for small-scale sequencing studies. However, many existing RNA-sequencing analysis frameworks are designed for transcriptome-wide analyses and require substantial computational resources. Here we present a lightweight and reproducible computational pipeline for targeted long-read transcriptomic profiling using Oxford Nanopore Technologies (ONT) cDNA sequencing data. The pipeline was evaluated using targeted long-read transcriptomic datasets generated from formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma samples previously classified as microsatellite instability—high (MSI-high) by PCR-based testing. Libraries were sequenced on the Oxford Nanopore MinION platform using R10.4.1 flow cells. Application of the workflow enabled rapid quantification of mismatch repair gene expression and detection of immune-related transcripts including CD8A, PDCD1, and HAVCR2 across multiplexed barcode samples. The pipeline performs targeted alignment of long-read sequencing data to a custom transcript reference panel using minimap2, followed by gene-level read counting and normalization using reads-per-million (RPM). Optional modules enable immune marker profiling, detection of reads aligning to multiple genes, exploratory variant analysis, and visualization of expression patterns. By combining simplicity, reproducibility, and minimal computational overhead, the present pipeline provides an accessible framework for targeted transcriptomic analysis of long-read sequencing data. It may facilitate adoption of ONT-based transcriptomic profiling in settings with restricted computational resources. Full article

Infectious diseases remain a major global health concern, with a growing burden of antimicrobial resistance and consequent higher mortality in the human population. Accurate bacterial identification is fundamental across clinical, veterinary, agricultural, and research settings, supporting effective diagnosis, antimicrobial stewardship, infection control, food safety, and environmental monitoring; however, conventional approaches are limited by time constraints, reduced sensitivity, and challenges in detecting fastidious or uncultivable organisms. This review provides a comprehensive overview of classical and advanced methods, including microscopy, culture, biochemical testing, immunological and serological assays, proteomic and spectroscopy-based techniques, and molecular approaches, such as polymerase chain reaction (PCR), digital PCR, DNA hybridization, 16S rRNA gene sequencing, whole-genome sequencing, and metagenomics. The integration of artificial intelligence has further enhanced analytical performance. Nevertheless, harmonization of bioinformatics frameworks remains essential, as variability in algorithm-defined cut-off values limits standardized implementation of whole-genome sequencing in routine laboratories. Emerging technologies, including CRISPR-based diagnostics and phage- and nanomaterial-based detection systems, offer promising alternatives. Overall, the integration of these approaches is expected to improve the accuracy, speed, and applicability of bacterial identification across diverse settings; however, these advances should be implemented cautiously, with standardization remaining a key priority alongside technological modernization. Full article

White pine blister rust disease (WPBR), caused by Cronartium ribicola, ranks among the most destructive pathogens of five-needle pines. We developed a hydroxynaphthol blue (HNB)-based Loop-mediated isothermal amplification (LAMP) assay enabling rapid, visual detection of C. ribicola directly following DNA extraction. LAMP primers targeting the internal transcribed spacer (ITS) region were designed and validated through in silico comparison with related Cronartium species and in vitro testing against sympatric forest fungi. The optimized 25 μL reaction contained 8.0 mM Mg²⁺, 1.0 mM dNTPs, and an inner-to-outer primer ratio of 8:1, with amplification conducted at 62 °C for 40 min. Positive amplification produced a distinctive color transition from purple to sky blue, enabling visual interpretation without instrumentation. Under the tested conditions, the assay achieved a detection limit of 460 ± 3.2 fg/μL genomic DNA—a 10-fold improvement over conventional PCR in concentration-based sensitivity. Assay applicability was evaluated using 211 field-collected Pinus armandii samples sourced from China. Detection efficiency varied significantly across tissue types. Symptomatic bark exhibited a substantially higher positive detection rate (68.97%, 95% CI: 49.2–84.7%) compared to needles from symptomatic trees (18.75%, 95% CI: 4.1–45.7%). Among asymptomatic samples, 3.75% of bark samples tested positive for C. ribicola DNA, whereas all needle samples were negative. Geographically, positive detections clustered at several discrete sampling sites in southwestern China, predominantly at elevated elevations. The established LAMP-HNB assay provides a rapid, visually interpretable diagnostic tool for early detection and quarantine monitoring of WPBR following DNA extraction. Beyond its practical utility, this assay establishes valuable baseline data for targeted disease surveillance in the context of evolving climate conditions. Full article