Search Results (515)

Background: Enterotoxigenic Bacteroides fragilis (ETBF) carries the bft toxin gene, which influences the host immune response and inflammatory pathways and promotes colorectal cancer (CRC). This study investigated the potential role of ETBF in CRC liver metastasis. Methods: We reviewed the records of 226 consecutive patients who underwent curative-intent (R0) resection of CRC liver metastases. ETBF DNA in fresh-frozen metastasis specimens was quantified using droplet digital PCR (ddPCR). Patients were grouped into very-low (≤80%; N = 178), low (80–90%; N = 24), and high (>90%; N = 24) ETBF-DNA groups. Three tissue cores per specimen were stained for CD8, CD4, CD20, FOXP3, CD68, and CD163, and immune-cell densities were measured digitally (cells/mm²). Results: ETBF DNA was detected in 219 of 226 lesions (96.9%). The densities of cytotoxic CD8⁺ T-cells, effector CD4⁺ T-cells, CD20⁺ B-cells, and CD163⁺ macrophages did not differ significantly by ETBF-DNA group (P_trend all > 0.12). FOXP3⁺ regulatory T-cells (Tregs) decreased (P_trend = 0.010), and CD68⁺ macrophages increased (P_trend = 0.020) as ETBF-DNA levels increased. ETBF-DNA levels in CRC liver metastases were not associated with disease-free survival or overall survival or serum C-reactive protein levels. Conclusions: ETBF was present in almost all CRC liver metastases. Higher ETBF levels were associated with a tumor-immune microenvironment enriched in CD68⁺ macrophages and deficient in FOXP3⁺ Tregs, suggesting that ETBF facilitates immune evasion without loss of effector lymphocytes. Although ETBF-DNA levels did not predict survival in this single-center cohort, the potential role of ETBF in immune remodeling and as a candidate biomarker and therapeutic target in metastatic CRC warrants further study. Full article

Talaromycosis caused by Talaromyces marneffei is a life-threatening mycosis in patients with acquired immunodeficiency syndrome (AIDS). The gold-standard diagnostic method relies on time-consuming cultures, which delay treatment and increase mortality. In this study, we developed a rapid and sensitive droplet digital PCR (ddPCR) assay targeting the internal transcribed spacer (ITS) gene for detecting T. marneffei and compared its performance with blood culture and quantitative PCR (qPCR) assays. The ddPCR assay had a detection limit of one copy/reaction, making it 10-fold more sensitive than qPCR. It demonstrated 100% specificity for T. marneffei, with no cross-reactivity to 15 other fungal pathogens, six bacterial pathogens, and plasma from 119 AIDS patients without talaromycosis. In 119 AIDS patients with talaromycosis, ddPCR exhibited better overall sensitivity (92.44%) than blood culture (86.55%) and qPCR (87.29%). The sensitivity of ddPCR was 97.8% (89/91) and 75% (21/28) in plasma collected before and after antifungal therapy, respectively. Moreover, fungal load measured by ddPCR negatively correlated with the time to blood culture positivity. Fungal loads in patients receiving antifungal therapy were significantly lower than those in untreated patients. These findings indicate that ddPCR facilitates rapid diagnosis of T. marneffei infection in AIDS patients and can assist clinicians in evaluating treatment efficacy by quantifying fungal load. Full article

Background/Objectives: Esophageal and esophagogastric junction adenocarcinoma (EADC-EGJA), which mainly develops from Barrett’s esophagus (BE), low-grade dysplasia (LGD), and high-grade dysplasia (HGD), has a poor prognosis and several unmet clinical needs, among which is the detection of minimal residual disease (MRD) after endoscopic/surgical resection. Long interspersed nuclear element-1 (LINE-1), a surrogate marker of global methylation, is considered an emerging biomarker for MRD monitoring. The aim of this study was to determine, by LINE-1 methylation analysis, at which carcinogenesis step global methylation is affected and whether this biomarker could be followed in longitudinal to monitor the disease behavior post-surgery. Methods: Cell-free DNA of 90 patients with non-dysplastic Barrett’s esophagus (NDBE), HGD/early EADC-EGJA, or locally advanced/advanced EADC-EGJA were analyzed for LINE-1 methylation, by Methylation-Sensitive Restriction Enzyme droplet digital PCR (MSRE-ddPCR). Twenty-six patients were longitudinally studied by repetitive blood sampling. Results: Global hypomethylation increased during carcinogenesis, with significant difference between locally advanced/advanced EADC-EGJA and NDBE patients (p = 0.028). Longitudinal cases confirmed the rareness of hypomethylation in NDBE cases. The majority of HGD/early EADC-EGJA and locally advanced/advanced EADC-EGJA patients showed methylation changes after resection according to clinical status. Conclusions: This study suggests that global hypomethylation occurs just prior to cancer invasiveness and that it is a promising biomarker to monitor MRD. Full article

In recent years, digital microfluidic biochips (DMFBs), based on microfluidic technology, have attracted attention as compact and flexible experimental devices. DMFBs are widely applied in biochemistry and medical fields, including point-of-care clinical diagnostics and PCR testing. Among them, micro electrode dot array (MEDA) biochips, composed of numerous microelectrodes, have overcome the limitations of conventional chips by enabling finer droplet manipulation and real-time sensing, thus significantly improving experimental efficiency. While various studies have been conducted to enhance the utilization of MEDA biochips, few have considered the shape-dependent velocity characteristics of droplets in routing. Moreover, methods that do take such characteristics into account often face significant challenges in solving time. This study proposes a fast droplet routing method for MEDA biochips that incorporates shape-dependent velocity characteristics by utilizing the distance information to the target cell. The experimental results demonstrate that the proposed method achieves approximately a 67.5% reduction in solving time compared to existing methods, without compromising solution quality. Full article

The BRAF V595E mutation analysis in canine urothelial carcinomas (UCs) has found its way into routine diagnostics, but no data analysis has been published until now. The present study aimed to estimate the distribution of age, sex, and breed in 8365 canine diagnostic samples submitted for BRAF mutation analysis during 2018–2024. The specimens included 8215 urine samples, 17 cytological, and 133 histopathological specimens, and were submitted in cases of suspected UC, to rule out UC, or for screening purposes. All samples were tested for the BRAF V595E mutation using droplet digital PCR (ddPCR). The data were statistically analysed and logistic regression models (Odds Ratio (OR)) were calculated. Compared to samples from mixed-breed dogs, the specimens from Scottish Terriers (OR: 4.21), Shetland Sheepdogs (OR: 2.65), Beagles (OR: 2.33), Fox Terriers (OR: 1.92), Staffordshire Bull Terriers (OR: 1.86), Magyar Vizslas (OR: 1.77), Chihuahuas (OR: 1.70), and West Highland White Terriers (OR: 1.43) had a significantly increased probability of the presence of BRAF mutation indicating UC. The youngest BRAF-positive dogs of these predisposed breeds (n = 4) were 5 years old. In conclusion, screening tests in predisposed breeds may be recommended from the age of 5 years. Full article

Accurate gene expression quantification using reverse transcription quantitative PCR (RT-qPCR) requires stable reference genes (RGs) for reliable normalization. However, few studies have systematically identified RGs suitable for simultaneous high salt, alkaline, and high-temperature conditions. This study addresses this gap by evaluating the stability of eight candidate RGs in the anaerobic halophilic alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF^T under combined salt, alkali, and thermal stresses. The stability of these candidate RGs was assessed using five statistical algorithms: Delta CT, geNorm, NormFinder, BestKeeper, and RefFinder. Results indicated that recA exhibited the highest expression stability across all tested conditions and proved adequate as a single RG for normalization in both RT-qPCR and droplet digital PCR (ddPCR) assays. Furthermore, recA alone or combined with other RGs (sigA, rsmH) effectively normalized the expression of seven stress-response genes (proX, opuAC, mnhE, nhaC, trkH, ducA, and pimT). This work represents the first systematic validation of RGs under polyextreme stress conditions, providing essential guidelines for future gene expression studies in extreme environments and aiding research on microbial adaptation mechanisms in halophilic, alkaliphilic, and thermophilic microorganisms. Full article

BRCA1 and BRCA2 are associated with advanced prostate cancer progression and poor prognosis. Copy number variants (CNVs) of these genes play a crucial role in guiding targeted treatments, particularly for patients receiving PARP inhibitors. However, CNV detection using multiplex ligation-dependent probe amplification (MLPA) is often limited by tumor heterogeneity, leading to ambiguous results. This study therefore aimed to evaluate BRCA1/2 CNVs in advanced prostate cancer patients using droplet digital PCR (ddPCR) and compare the results with MLPA. DNA from 11 advanced prostate cancer tissues was analyzed using both methods, in parallel with four cell lines and seven healthy volunteers. Our findings revealed that ddPCR effectively classified normal CNV groups—including normal control cell lines, healthy volunteers, and samples with normal MLPA final ratios—from deletion groups, which included deletion control cell lines, samples with deletion final ratios from MLPA, and cases with previously ambiguous results. Interestingly, two cases involving BRCA1 and one case involving BRCA2 exhibited ambiguous results using MLPA; however, ddPCR enabled more precise classification by applying the Youden Index from ROC analysis and identifying optimal cutoff values of 1.35 for BRCA1 and 1.55 for BRCA2. These optimal thresholds allowed ddPCR to effectively reclassify the ambiguous MLPA cases into the deletion group. Overall, ddPCR could offer a more sensitive and reliable approach for CNV detection in heterogeneous tissue samples and demonstrates strong potential as a biomarker tool for guiding targeted therapy in advanced prostate cancer patients. However, further validation in larger cohorts is necessary to optimize cutoff precision, confirm diagnostic performance, and evaluate the full clinical utility of ddPCR. Full article

Background/Objectives: Antibiotic resistance presents an urgent public health threat. By developing a streamlined and effective method for studying bacteriophage induction, this research marks a step further in understanding how antibiotic-resistant genes might spread across different environments. This knowledge is essential for creating strategies to reduce the spread of antimicrobial resistance (AMR), particularly from a One Health perspective. In this study, we develop and validate a Green Fluorescent Protein (GFP)-based method as a proxy for bacteriophage induction. This method screens compounds for their potential to promote bacteriophage induction. Methods: This study utilized a recA-GFP construct in Escherichia coli to measure fluorescence as an indicator of SOS response activation. The experiments involved treating E. coli cultures with varying concentrations of the DNA-damaging chemical mitomycin C and measuring fluorescence over time. Additionally, droplet digital PCR (ddPCR) quantified bacteriophage induction in a lambda phage-carrying E. coli strain, allowing for correlation analysis between the two methods. Results: The recA-driven SOS response depended on both dose and time, with increasing concentrations of mitomycin C leading to higher fluorescence. ddPCR analysis confirmed that mitomycin C induced prophage activation, with gene ratios increasing at higher drug concentrations over time. A strong Spearman correlation (>0.7) was noted between fluorescence and ddPCR results at elevated concentrations and relevant time points, indicating the validity of the GFP-based model as a proxy for bacteriophage induction. Conclusions: The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based E. coli model is a reliable, cost-effective, and efficient tool for studying phage induction and its potential role in AMR spread. This method could facilitate the screening of environmental samples and specific drugs to evaluate their impact on bacteriophage induction, which opens the door for applications such as screening for antibiotic resistance dissemination. Full article

Indoors, the infection risk of diseases transmitted through the airborne route is estimated from indoor carbon dioxide (CO₂) levels. However, the approaches to assess this risk do not account for the airborne concentration of pathogens, among other limitations. In this study, we analyzed the relationship between airborne SARS-CoV-2 levels and environmental parameters. Bioaerosols were sampled (n = 40) in hospital corridors of two wards differing in the COVID-19 severity of the admitted patients. SARS-CoV-2 levels were quantified using droplet digital PCR. SARS-CoV-2 was detected in 60% of the total air samples. The ward where the mildly ill patients were admitted had a higher occupancy, transit of people in the corridor, and CO₂ levels, but there were no significant differences in SARS-CoV-2 detection between wards. The mean CO₂ concentration in the positive samples was 569 ± 35.6 ppm. Considering all samples, the CO₂ levels in the corridor were positively correlated with patient door openings but inversely correlated with SARS-CoV-2 levels. In conclusion, airborne SARS-CoV-2 can be detected indoors with optimal ventilation, and its levels do not scale with CO₂ concentration in hospital corridors. Therefore, CO₂ assessment should not be interpreted as a surrogate of airborne viral presence in all indoor spaces. Full article

Background/Objectives: Cxbladder^® Triage Plus is a multimodal urinary biomarker assay that combines reverse transcription-quantitative analysis of five mRNA targets and droplet-digital polymerase chain reaction (ddPCR) analysis of six DNA single-nucleotide variants (SNVs) from two genes (fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT)) to provide risk stratification for urothelial carcinoma (UC) in patients with hematuria. This study evaluated the analytical validity of Triage Plus. Methods: The development dataset used urine samples from patients with microhematuria or gross hematuria that were previously stabilized with Cxbladder solution. Triage Plus was evaluated for predicted performance, analytical criteria (linearity, sensitivity, specificity, accuracy, and precision), extraction efficiency, and inter-laboratory reproducibility. Results: The development dataset included 987 hematuria samples. Compared with cystoscopy (standard of care), Triage Plus had a predicted sensitivity of 93.6%, specificity of 90.8%, positive predictive value (PPV) of 46.5%, negative predictive value of 99.4%, and test-negative rate of 84.1% (score threshold 0.15); the PPV increased to 74.6% for the 0.54 score threshold. For the individual FGFR3 and TERT SNVs, the limit of detection (analytical sensitivity) was a mutant-to-wild type DNA ratio of 1:440–1:1250 copies/mL. Intra- and inter-assay variance was low, while extraction efficiency was high. All other pre-specified analytical criteria (linearity, specificity, and accuracy) were met. Triage Plus showed good reproducibility (87.9% concordance between laboratories). Conclusions: Cxbladder Triage Plus accurately and reproducibly detected FGFR3 and TERT SNVs and, in combination with mRNA expression, provides a non-invasive, highly sensitive, and reproducible tool that aids in risk stratification of patients with hematuria. Full article

Torquetenovirus (TTV) is a ubiquitous, non-pathogenic DNA virus that has been suggested as a biomarker of immune competence, with the viral load correlating with the level of immunosuppression. However, by detecting non-intact viral particles, standard PCR-based quantification may overestimate the TTV viremia. To improve the clinical relevance of TTV quantification, in this study, we investigated the use of PMAxx™, a virion viability dye that selectively blocks the amplification of compromised virions. Serum samples from 10 Hepatitis C Virus-positive (HCV+) individuals, 81 liver transplant recipients (LTRs), and 40 people with HIV (PWH) were treated with PMAxx™ and analyzed for TTV DNA loads by digital droplet PCR (ddPCR). Furthermore, anti-SARS-CoV-2 IgG levels and neutralizing antibody (nAbs) titers were measured post-COVID-19 vaccination. Using ddPCR, the PMAxx™ treatment significantly reduced the TTV DNA levels in all the groups (mean reduction: 0.66 Log copies/mL), indicating the abundant presence of non-intact, circulating viral genomes. However, correlations between TTV DNA and SARS-CoV-2 IgG or nAbs were weak or absent in both PMAxx™-treated and untreated samples. These findings suggest that while PMAxx™ enhanced the specificity of TTV quantification, it did not improve the predictive value of TTV viremia at assessing vaccine-induced humoral responses. Full article

Usher syndrome, a rare genetic disorder causing both hearing and vision loss, presents significant diagnostic and therapeutic challenges due to its complex genetic basis. The identification of reliable biomarkers for early detection and intervention is crucial for improving patient outcomes. In this study, we present a machine learning-based hybrid sequential feature selection approach to identify key mRNA biomarkers associated with Usher syndrome. Beginning with a dataset of 42,334 mRNA features, our approach successfully reduced dimensionality and identified 58 top mRNA biomarkers that distinguish Usher syndrome from control samples. We employed a combination of feature selection techniques, including variance thresholding, recursive feature elimination, and Lasso regression, integrated within a nested cross-validation framework. The selected biomarkers were further validated using multiple machine learning models, including Logistic Regression, Random Forest, and Support Vector Machines, demonstrating robust classification performance. To assess the biological relevance of the computationally identified mRNA biomarkers, we experimentally validated candidates from the top 10 selected mRNAs using droplet digital PCR (ddPCR). The ddPCR results were consistent with expression patterns observed in the integrated transcriptomic metadata, reinforcing the credibility of our machine learning-driven biomarker discovery framework. Our findings highlight the potential of machine learning-driven biomarker discovery to enhance the detection of Usher syndrome. Full article

Toxoplasma gondii, Giardia intestinalis, and Cryptosporidium spp. are common pathogens that contaminate water and food. They can pose serious health risks, especially to vulnerable groups like immunocompromised individuals, pregnant women, young children, and aging people. An all-encompassing approach to minimizing transmission involves identifying effective techniques for detecting, treating, and preventing protozoan parasites. This study confirmed the effectiveness of a Droplet Digital Reverse Transcription Polymerase Chain Reaction (dd RT-PCR) method for quickly and accurately identifying Toxoplasma gondii, Giardia intestinalis, and Cryptosporidium species in honeybees, honey, and pollen by using ISO 17468 and ISO 16140 standard guidelines. The study evaluated honeybee (n = 16), honey (n = 12), and pollen (n = 8) samples collected from various apiaries in Southern Italy between June and September 2023. The results showed that honeybees, honey, and pollen can be considered bioindicators of infections by T. gondii, G. intestinalis, and Cryptosporidium spp. Furthermore, pollen, along with honey to a lesser degree, can serve as significant indicators for evaluating food safety. Therefore, it is essential to monitor their quality and purity due to environmental influences. Full article

Hereditary α-tryptasemia (HαT)—a genetic trait caused by increased α-tryptase-encoding typtase alpha/beta-1 (TPSAB1) copy number—is associated with adult mastocytosis. The primary objective was to assess the association between α-tryptase and pediatric mastocytosis. We also want to evaluate whether the KIT p.D816V mutation in peripheral blood leukocytes (PBLs) reliably predicts systemic mastocytosis (SM) in children. A prospective cohort of 68 children from a referral center in Slovenia with cutaneous mastocytosis (CM) underwent tryptase genotyping by droplet digital PCR and examination for KIT p.D816V in PBL using a sensitive PCR test. A significant majority of patients (57 of 68; [83.8%]) had at least one α-tryptase-encoding gene; none had HαT. 7 of the 68 (10.3%) who were positive for KIT p.D816V in PBL, one fulfilled diagnostic criteria for indolent SM, and another was diagnosed with monoclonal mast cell activation syndrome. One of those individuals had an increased basal serum tryptase (BST) level (14.5 ng/mL). We found a high presence of germline α-tryptase in children with CM, but not HαT. By employing sensitive examination for KIT p.D816V in PBL, in combination with clinical data and other examinations, our study suggests that KIT p.D816V in PBL may indicate systemic disease in children with CM. Full article

Olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi, severely impacts olive tree yield and oil quality. Early and accurate detection of the bacterium’s presence, particularly in asymptomatic plants, is crucial for effective disease management. This study aimed to develop an improved protocol for processing plant samples and adapting quantitative PCR to droplet digital PCR (ddPCR). For this purpose, four plant preparations—EW (external washing), PELLET (bacterial concentration), and enrichment in liquid media for 24 or 48 h (24hE, 48hE)—were tested using spiked samples. The ddPCR was set up and compared with qPCR to evaluate analytical sensitivity and specificity. Additionally, field samples from symptomatic and asymptomatic olive orchards were tested to evaluate the performance of the selected methods in naturally infected plants. ddPCR showed higher sensitivity than qPCR, particularly with the PELLET and 24hE preparations. The PELLET from the spiked sample preparation achieved a limit of detection of 10 CFU/mL for both molecular tests. The ddPCR, combined with the PELLET preparation, offers a highly sensitive and reliable tool for detecting P. savastanoi pv. savastanoi in asymptomatic olive material. This protocol shows great potential for improving early bacterial detection and disease prevention, thus aiding control strategies in nurseries and olive orchards, and supporting the production of certified plant propagation material. Full article