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Background and Objectives Materials and Methods Results Conclusion To test the performance of a new droplet digital polymerase chain reaction (ddPCR) non‐invasive foetal blood group and platelet antigen genotyping assay in the setting of a Dutch reference laboratory for foetal blood group and platelet antigen genotyping. Our population comprised 229 consecutive alloimmunized pregnant women who presented between April 2022 and March 2023 with 250 requests for non‐invasive foetal RHD, RHE, RHc, RHC, K1, HPA‐1a or HPA‐5b blood group and platelet antigen genotyping.Samples were genotyped for blood group and platelet antigen alleles along with methylated RASSF1a (mRASSF1a) and sex‐determining region of Y (SRY) and DYS14 as positive foetal controls. Negative blood group and platelet antigen results were issued only when foetal controls were positive; otherwise, such samples were classified as inconclusive.The assay achieved a success rate of 98.4% (246 of 250) because one case was lost to follow‐up, one case was solved with quantitative polymerase chain reaction (qPCR) and one case precluded foetal typing due to RHD variant mothers. Only 10 cases needed a second sample and one case a third for a valid final result. We identified 116 maternal–foetal blood group and platelet antigen incompatibilities.Clinical non‐invasive foetal blood group and platelet antigen typing of alloimmunized pregnant women via ddPCR is successful and represents an improvement over qPCR because of the addition of a foetal control and because ddPCR circumvents potential interference from maternal cell‐free DNA (cfDNA) background for foetal HPA‐1 and K1. [ABSTRACT FROM AUTHOR]

Background: The introduction of adjuvant therapies for patients with resected cutaneous melanoma (CM) has increased the need for sensitive biomarkers for risk stratification and disease monitoring. This study aims to investigate the utility of circulating tumor DNA (ctDNA) assessment in predicting and reflecting disease status during adjuvant therapy. Methods: We enrolled 32 patients with resected BRAF-mutated stage III CM receiving adjuvant targeted therapy or immunotherapy. Plasma samples of patients were collected at the baseline (treatment initiation) and during the therapy, and BRAF-mutated ctDNA was quantified by droplet digital PCR (ddPCR). Results: Baseline ctDNA was detected in 11/32 (34.4%) patients and predicted postoperative high risk of relapse [HR 3.79, 95% CI 1.20–12.00, p = 0.023]. The three-year overall survival (OS) rate was 54.6% (95% CI 22.9–77.9) versus 95% (95% CI 69.5–99.3) in ctDNA-positive and negative groups, respectively, with significantly worse OS for ctDNA-positive patients [HR 7.92, 95% CI 1.56–40.36, p = 0.013]. Among the baseline ctDNA-positive group (high-risk patients), longitudinal ctDNA detection during adjuvant therapy reflected the clinical outcomes. Only non-relapsing patients cleared their plasma ctDNA by the end of the treatment, while persistent ctDNA detection provided early evidence of disease recurrence. Conclusions: ctDNA detection shows promising results in the post-operative setting for identifying cutaneous melanoma patients at the highest risk of relapse and for real-time monitoring of patients' clinical status and treatment response. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs) are a group of nanoscale cell-derived membranous structures secreted by all cell types, containing molecular cargoes involved in intercellular communication. EVs can be used to mimic "nature's delivery system" to transport nucleic acids, peptides, lipids, and metabolites to target recipient cells. EVs offer a range of advantages over traditional synthetic carriers, thus paving the way for innovative drug delivery approaches that can be used in different diseases, including cancer. Here, by using breast cancer (BC) cells treated with the multi-kinase inhibitor sorafenib, we generated EVs enriched in specific non-coding RNAs (miR-23b-3p, miR-126-3p, and the long ncRNA GAS5) and investigated their potential impact on the aggressive properties of the BC in vitro and in vivo using zebrafish. Methods: EVs were collected from 4 different BC cell lines (HCC1937, MDA-MB-231, MCF-7, and MDA-MB-453) and characterized by western blotting, transmission electron microscopy and nanoparticle tracking analysis. Levels of encapsulated miR-23b-3p, miR-126-3p, and GAS5 were quantified by ddPCR. The role of the EVs as carriers of ncRNAs in vivo was established by injecting MDA-MB-231 and MDA-MB-453 cells into zebrafish embryos followed by EV-based treatment of the xenografts with EVs rich in miR-23b-3p, miR-126-3p and GAS5. Results: ddPCR analysis revealed elevated levels of miR-23b-3p, miR-126-3p, and GAS5, encapsulated in the EVs released by the aforementioned cell lines, following sorafenib treatment. The use of EVs as carriers of these specific ncRNAs in the treatment of BC cells resulted in a significant increase in the expression levels of the three ncRNAs along with the inhibition of cellular proliferation in vitro. In vivo experiments demonstrated a remarkable reduction of xenograft tumor area, suppression of angiogenesis, and decreased number of micrometastasis in the tails after administration of EVs enriched with these ncRNAs. Conclusions: Our study demonstrated that sorafenib-induced EVs, enriched with specific tumor-suppressor ncRNAs, can effectively inhibit the aggressive BC characteristics in vitro and in vivo. Our findings indicate an alternative way to enrich EVs with specific tumor-suppressor ncRNAs by treating the cells with an anticancer drug and support the development of new potential experimental molecular approaches to target the aggressive properties of cancer cells. [ABSTRACT FROM AUTHOR]

Efficient early pathogen detection, before symptom apparition, is crucial for optimizing disease management. In barley, the fungal pathogen Pyrenophora teres is the causative agent of net blotch disease, which exists in two forms: P. teres f. sp. teres (Ptt), causing net-form of net blotch (NTNB), and P. teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (STNB). In this study, we developed primers and a TaqMan probe to detect both Ptt and Ptm. A comprehensive k-mer based analysis was performed across a collection of P. teres genomes to identify the conserved regions that had potential as universal genetic markers. These regions were then analyzed for their prevalence and copy number across diverse Moroccan P. teres strains, using both a k-mer analysis for sequence identification and a phylogenetic assessment to establish genetic relatedness. The designed primer-probe set was successfully validated through qPCR, and early disease detection, prior to symptom development, was achieved using ddPCR. The k-mer analysis performed across the available P. teres genomes suggests the potential for these sequences to serve as universal markers for P. teres, transcending environmental variations. [ABSTRACT FROM AUTHOR]

Background: The screening of TERT promoter (TERTp) mutations is essential in cancer research and diagnostics, due to its prevalence in tumours associated with low self-renewal rates. TERTmonitor is a diagnosis kit primarily designed for real-time qPCR qualitative detection of −124C>T and −146C>T TERTp mutations, which are highly prevalent in several malignancies, particularly in bladder carcinoma. Objective: This study aims to investigate TERTmonitor performance in droplet digital PCR (ddPCR) in urine samples from bladder cancer patients. Methods: A total of 45 urine samples were examined by real-time qPCR and ddPCR techniques, and their performances were compared. Results: TERTmonitor had similar performance in both real-time qPCR and ddPCR platforms. Specifically, the methods exhibited a concordance rate of 95.45% and 90% for −124C>T and −146C>T mutations, respectively. Importantly, an enhanced sensitivity in certain scenarios was exhibited by ddPCR when compared to real-time qPCR, detecting mutations that the latter failed to identify in approximately 4.55% and 10% of the samples for −124C>T and −146C>T mutations, respectively. This enhanced sensitivity of ddPCR was particularly evident in samples with low-frequency mutations. Conclusions: The findings highlight the usefulness of TERTmonitor for cancer surveillance either in real-time qPCR or ddPCR platforms. [ABSTRACT FROM AUTHOR]

Simple Summary: Fungicide resistance is the natural and inheritable adaptation of pathogens to survive treatment with a phytosanitary product that would normally provide effective control. Plasmopara viticola, the causal agent of Grapevine Downy Mildew (GDM), is an important pathogen in vineyards, in which resistance to Carboxylic Acid Amide (CAA) fungicides has been observed and reported. Behind this resistance, there are two single-point substitutions of the cellulose synthase gene: G1105S and G1105V. In this article, we developed a droplet digital polymerase chain reaction (ddPCR) protocol for the quantification of the mutations conferring the resistance. The ddPCR protocol precisely determined allele frequencies in four fields where P. viticola bulk samples were collected. Plasmopara viticola is the causal agent of Grapevine Downy Mildew (GDM), which is a devastating disease of grapevines in humid temperate regions. The most employed method for protecting grapevines against GDM is the application of chemical fungicides. In Spain, Carboxylic Acid Amides (CAAs) are a fungicide group currently utilized in GDM control. In P. viticola, resistance to CAAs is conferred by G1105S and G1105V mutations in the CesA3 gene. Droplet digital polymerase chain reaction (ddPCR) is an innovative technique that combines PCR and droplet microfluidics to disperse the sample into thousands of water-in-oil droplets in which an amplification reaction is individually performed. In this study, we set up a ddPCR protocol to quantify S1105 and V1105 mutations conferring resistance to CAAs in P. viticola. The optimal PCR conditions were established, and the sensitivity and precision of the protocol were assessed. Four P. viticola populations coming from commercial vineyards in northern Spain were analyzed, and different allele frequencies were found in the analyzed samples corresponding to the different fungicide management strategies, ranging from 7.72% to 100%. Knowing the level of mutated alleles allows for designing resistance management strategies suited for each location. This suggests that similar ddPCR assays could be developed for studying mutations implicated in fungicide resistance in other fungicide groups and plant pathogens. [ABSTRACT FROM AUTHOR]

According to World Health Organization (WHO), antimicrobial resistance (AMR) is currently one of the world's top 10 health threats, causing infections to become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. Accurate surveillance is a key component in the fight against AMR. Wastewater is progressively becoming a new player in AMR surveillance, with the promise of a cost‐effective real‐time tracking of global AMR profiles in specific regions. One of the most useful analytical methods for wastewater surveillance is currently based on real‐time PCR (qPCR) and digital droplet PCR (ddPCR) technologies. As stated in the EU Wastewater Treatment Directive proposal, methodological standardization, including a workflow for method development and validation, will play a crucial role in global monitoring of AMR in wastewater. However, according to our knowledge, there are currently no qPCR and ddPCR methods for AMR surveillance available that have been validated according to international standard performance criteria. Therefore, this study proposes a workflow for the development and validation of PCR‐based methods for a harmonized and global AMR surveillance, including the construction of specific sequence databases and microbial collections for an efficient method development and method specificity evaluation. Following this strategy, we have developed and validated four duplex ddPCR methods responding to international standard performance criteria, focusing on seven AMR genes (ARG's), including extended spectrum beta‐lactam (blaCTX‐M), carbapenem (blaKPC‐2/3), tetracycline (tet(M)), erythromycin (erm(B)), vancomycin (vanA), sulfonamide (sul2), and aminoglycoside (aac(3)‐IV), as well as one indicator of antibiotic (multi‐) resistance and horizontal gene transfer, named the class I integron (intl1). The performance of these ddPCR methods was successfully assessed for their specificity, as no false‐positive and false‐negative results were observed. These ddPCR methods were also considered to be highly sensitive as showing a limit of detection below 25 copies of the targets. In addition, their applicability was confirmed using 14 wastewater samples collected from two Belgian water resource recovery facilities. The proposed study represents therefore a step forward to reinforce method harmonization in the context of the global AMR surveillance in wastewater. Practitioner Points: In the context of wastewater surveillance, no PCR‐based methods for global AMR monitoring are currently validated according to international standards.Consequently, we propose a workflow to develop and validate PCR‐based methods for a harmonized and global AMR surveillance.This workflow resulted here in four duplex ddPCR methods targeting seven ARGs and one general indicator for mobilizable resistance genes.The applicability of these validated ddPCR methods was confirmed on 14 wastewater samples from two Belgian water resource recovery facilities. [ABSTRACT FROM AUTHOR]

Background: The role of microbiome, particularly Fusobacterium nucleatum (Fn), in periodontal disease and oral squamous cell carcinoma (OSCC) has been recently explored. This study aimed to evaluate the Fn presence and its levels in oral rinse samples from Brazilian OSCC patients and healthy individuals and its association with sociodemographic, clinical, and oral health features. Methods: In this case–control study, 80 participants were included, 31 OSCC patients and 49 individuals without a cancer history. Clinical data were collected, and an oral exam was done on a subset of the cohort. Fn levels were evaluated by droplet digital PCR (ddPCR) in oral rinse samples and were categorized as Fn‐high or Fn‐low based on the median number of copies per reaction. Results: OSCC patients showed higher levels of Fn (68%, p = 0.03) than controls, and all OSCC cases were diagnosed with periodontal disease (100%, p = 1.0). In the univariate analysis, Fn‐high level was more frequently present in OSCC cases compared to controls (p = 0.01). It was also observed that Fn‐high level OSCC cases were significantly associated with self‐reported non‐white ethnicity (71.4%, p = 0.01) and had more infiltrative lesions (57.1%, p = 0.02) than Fn‐low OSCC cases. Fn‐high levels in oral rinse samples, were significantly more prevalent among OSCC than in controls. Conclusions: In OSCC patients, Fn‐high levels were associated with non‐white ethnicity and lesions with infiltrative clinical aspects. Among OSCC cases, all had periodontal disease. [ABSTRACT FROM AUTHOR]

Background/Objectives: Sarcomas are a heterogeneous group of cancers, many with high rates of recurrence and metastasis, leading to significant morbidity and mortality. Due to a lack of early diagnostic biomarkers, by the time recurrent disease can be clinically detected, it is often extensive and difficult to treat. Here, we sought to investigate methods of detecting ctDNA in sarcoma patient plasma to potentially monitor disease recurrence, progression, and response to treatment. Methods: Whole-exome sequencing of matched tumor and blood samples revealed patient-specific mutations, which were used to develop personalized assays to detect ctDNA in patient plasma. Since ctDNA is present in extremely low quantities, detection requires highly sensitive methodologies. Droplet digital PCR is highly sensitive; however, it is limited in that it can only be used to target one tumor variant at a time. Therefore, a protocol combining multiplex PCR and targeted amplicon sequencing was developed. Results: ddPCR was successfully able to detect tumor-specific mutations in plasma, confirming the presence of ctDNA in sarcoma patients. Multiplex PCR followed by amplicon sequencing was able to detect multiple tumor variants simultaneously, although it was not as sensitive as ddPCR. Additionally, ctDNA was detected in patient plasma collected at two different time points. Conclusions: This work demonstrates that although there is a lack of recurrent biomarkers, personalized assays detecting ctDNA have the potential to be used to monitor disease progression in sarcoma. [ABSTRACT FROM AUTHOR]

Simple Summary: Molecular profiling of cancer is used to examine for specific mutations or proteins and is becoming increasingly important in oncology. An example of such a mutation is the altered BRAF gene and the resulting changed BRAF protein, which is already known to play a role in urinary bladder and prostatic carcinomas in dogs. We assumed that the mutant BRAFV595E protein should be considered in other canine carcinomas and tested 227 samples of 11 different carcinoma origins (anal sac (n = 23), intestine (n = 21), liver (n = 21), lungs (n = 19), mammary gland (n = 20), nasal cavity (n = 21), oral epithelium (n = 18), ovary (n = 20), prostate (n = 21), thyroid gland (n = 21), urinary bladder (n = 22)). We used immunohistochemistry with two different primary antibodies, each binding to the altered BRAF protein, and confirmed our results with droplet digital PCR (ddPCR). Among all the tested canine carcinomas, we found BRAF-mutated tumours in the prostate (16/21), the urinary bladder (17/22), and the oral cavity epithelium (4/18), while other carcinomas tested negative. Our findings showed that both antibodies are dependable tools for detecting the BRAFV595E mutation in canine carcinomas. Detecting BRAF mutations is important for applying future therapeutic approaches, including BRAF inhibitors. Alterations of the BRAF gene and the resulting changes in the BRAF protein are one example of molecular cancer profiling in humans and dogs. We tested 227 samples of canine carcinomas from different anatomical sites (anal sac (n = 23), intestine (n = 21), liver (n = 21), lungs (n = 19), mammary gland (n = 20), nasal cavity (n = 21), oral epithelium (n = 18), ovary (n = 20), prostate (n = 21), thyroid gland (n = 21), urinary bladder (n = 22)) with two commercially available primary anti-BRAFV600E antibodies (VE1 Ventana, VE1 Abcam). The immunohistochemical results were confirmed with droplet digital PCR (ddPCR). BRAFV595E-mutated cases were found in canine prostatic (16/21), urothelial (17/22), and oral squamous cell carcinomas (4/18), while other carcinoma types tested negative. Both antibodies showed consistent results, with intracytoplasmic immunolabeling of tumour cells, making them reliable tools for detecting the BRAFV595E mutation in canine carcinomas. In conclusion, identifying BRAF mutations from biopsy material offers a valuable opportunity to enhance cancer treatment strategies (BRAF inhibitors) in canine urothelial carcinomas, prostatic carcinomas, and oral squamous cell carcinomas. [ABSTRACT FROM AUTHOR]

Detecting EGFR mutations in plasma using droplet digital PCR (ddPCR) assay offers a promising diagnostic tool for lung cancer patients. The performance of plasma-based ddPCR assay relative to traditional EGFR mutation testing in tissue biopsies among Asian patients with suspected lung cancer remains underexplored. Consecutive patients admitted for diagnostic workup for suspected lung cancer were recruited. Peripheral blood samples were collected on the same day of tissue biopsies. Tissue samples were subjected to EGFR mutation analysis via real-time PCR, whereas plasma samples were processed for ddPCR assay to evaluate for EGFR mutation status. The tissue re-biopsy rate was 43.8% while 0.7% of patients failed blood taking. Despite repeat biopsy, 15.2% of patients could not achieve histological diagnosis. Of the 202 patients newly diagnosed with lung cancer, EGFR mutations were detected in 13.4% of plasma samples, compared to 44.3% in tissue samples. Plasma ddPCR for EGFR mutations detection were barely detectable in stages I and II non-small cell lung cancer (NSCLC), but the sensitivity was 25.0%, 56.3%, and 75.0% in stages III, IVA, and IVB NSCLC, respectively. Plasma EGFR mutations were highly specific among all stages of lung cancer. Concordance rates of plasma ddPCR assay also rose with more advanced stages, recorded at 41.9% for stages I and II, 71.9% for stage III, 86.3% for stage IV. In stage IV lung cancer, the false negative rate for the plasma ddPCR assay was 34.4%, whereas that for the tissue testing was 19.2% due to insufficient tissue samples. Plasma-based EGFR genotyping using ddPCR is a non-invasive method that offers early diagnosis and serves as a valuable adjunct to tissue-based testing for patients with advanced-stage lung cancer. However, its usefulness is limited in the context of early-stage lung cancer, indicating a need for further research to improve its accuracy in these patients. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited survival. Curative opportunities are only available for patients with resectable cancer. Palliative chemotherapy is the current standard of care for unresectable tumors. Numerous efforts have been made to investigate new therapeutic strategies for PDAC. Immunotherapy has been found to be effective in treating tumors with high microsatellite instability (MSI-H), including PDAC. The ability of the Endoscopic Ultrasound Fine Needle Biopsy (EUS-FNB) to reliably collect tissue could enhance new personalized treatment by permitting genomic alterations analysis. The aim of this study was to investigate the feasibility of obtaining adequate DNA for molecular analysis from EUS-FNB formalin-fixed-paraffin-embedded (FFPE) specimens. For this purpose, FFPE-DNA obtained from 43 PDAC archival samples was evaluated to verify adequacy in terms of quantity and quality and was tested to evaluate MSI-H status by droplet digital PCR (ddPCR). All samples were suitable for ddPCR analysis. Unlike the 1–2% MSI-H frequency found with traditional techniques, ddPCR detected this phenotype in 16.28% of cases. This study suggests the ddPCR ability to identify MSI-H phenotype, with the possibility of improving the selection of patients who may benefit from immunotherapy and who would be excluded by performing traditional diagnostic methods. [ABSTRACT FROM AUTHOR]

The isolation of circulating tumoral DNA (ctDNA) present in the bloodstream brings about the opportunity to detect genomic aberrations from the tumor of origin. However, the low amounts of ctDNA present in liquid biopsy samples makes the development of highly sensitive techniques necessary to detect targetable mutations for the diagnosis, prognosis, and monitoring of cancer patients. Here, we employ standard genomic DNA (gDNA) and eight liquid biopsy samples from different cancer patients to examine the newly described CRISPR-Cas13a-based technology in the detection of the BRAF p.V600E actionable point mutation and appraise its diagnostic capacity with two PCR-based techniques: quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR). Regardless of its lower specificity compared to the qPCR and ddPCR techniques, the CRISPR-Cas13a-guided complex was able to detect inputs as low as 10 pM. Even though the PCR-based techniques have similar target limits of detection (LoDs), only the ddPCR achieved a 0.1% variant allele frequency (VAF) detection with elevated reproducibility, thus standing out as the most powerful and suitable tool for clinical diagnosis purposes. Our results also demonstrate how the CRISPR-Cas13a can detect low amounts of the target of interest, but its base-pair specificity failed in the detection of actionable point mutations at a low VAF; therefore, the ddPCR is still the most powerful and suitable technique for these purposes. [ABSTRACT FROM AUTHOR]

Background: Cytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins. Methods: Given the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle. Results: In affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs. Conclusions: Our study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines. [ABSTRACT FROM AUTHOR]

Background: Melanoma is the most aggressive skin cancer with ability to recur also after early‐stage tumor surgery. The aim was to identify early‐stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. Methods: Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre‐amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. Results: The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild‐type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). Conclusion: We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. Significance: There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early‐stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre‐ and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage. [ABSTRACT FROM AUTHOR]

Societal Impact Statement Summary The use of chemical plant protection products must be reduced to promote sustainability in food production. One possible alternative is biological control agents (BCAs), but their efficacy under commercial conditions does not always reach the standard of chemical control agents. Previously, light has been found to induce mechanisms in bacterial BCAs that can affect their distribution and establishment. This could promote BCA efficacy. We looked into how monochromatic and polychromatic (which is what growers use) light treatments affected the occurrence of three BCAs post‐application. By combining two non‐chemical methods: a biological (BCA) and a cultural (light) control method, this offers a new integrated pest control strategy. The dynamics and functionality of beneficial and non‐beneficial, non‐phototrophic bacteria can be influenced by light quality. We investigated if light could aid the survival of three bacterial biological control agents (BCAs; Bacillus amyloliquefaciens DSM7, Pseudomonas chlororaphis 50083 and Streptomyces griseoviridis CBS904.68) in the canopy of greenhouse‐grown tomatoes at four light treatments. Tomato plants were exposed to 50 μmol m−2 s−1 of either polychromatic light (white) or monochromatic light (blue: 420 nm, green: 530 nm and red: 660 nm) using DYNA LED lamps for a total of 48 h post foliar application of the BCAs. Leaves were harvested from two levels in the canopy at the top and middle of each plant at 0, 2, 4, 8, 12, 24 and 48 h post inoculation. The occurrences of the BCAs were quantified by plate count and droplet digital PCR (ddPCR). S. griseoviridis persisted under most treatments, whereas P. chlororaphis and B. amyloliquefaciens preferred the polychromatic and green light treatments as depicted by the viable count analyses. Significant differences between the DNA and cDNA concentrations were only noted for P. chlororaphis, with prominent wavelength effects. Light exposure dose, placement in the canopy and wavelength were found to be decisive factors for BCA re‐isolation, indicating that they have different optimal light environments. [ABSTRACT FROM AUTHOR]

To date, the increased awareness of the impact of microbes on human health has promoted scientific interest in microbiome studies for diagnostic and therapeutic purposes, revealing correlations between specific taxa and cancer. In particular, numerous species of Porphyromonas have been associated with several types of tumors. Previously, we studied the urobiome using Next-Generation Sequencing (NGS), and found an increase in Porphyromonas somerae in first morning urine of subjects affected by bladder cancer (BCa). Here, we aimed to confirm the presence of P. somerae in BCa patients by using droplet digital Polymerase Chain Reaction (ddPCR), testing a cohort of 102 male subjects over 50 years. Our findings showed a significant increase in P. somerae in the urine of the BCa group within both ddPCR and NGS, and a correlation between the two methods was observed at a statistical level. Moreover, P. somerae's identification with ddPCR confirmed a significant association between this bacterium and the presence of BCa, highlighting its potential role as a biomarker. This allows us to propose the ddPCR as a suitable method for first-stage BCa screening and follow-up. [ABSTRACT FROM AUTHOR]

Extrachromosomal circular DNA (eccDNA) has recently gained increasing attention due to its significant role in cancer and other pathophysiologic states. The majority of circular DNAs detected by Circle-seq are small-size eccDNAs with enigmatic functions. One major technical hurdle is to synthesize eccDNA for functional identification. Here, we describe CAES (Circle-seq based Artificial EccDNA Synthesis), a promising and reliable method for artificial eccDNA synthesis. Eight eccDNAs carrying different microRNA genes (eccMIR) found in gastric cancer tissues, ranging from 329 bp to 2189 bp in size, were created utilizing the CAES method. Exonuclease V and single restriction-endonuclease digestion identified the circular structure of synthetic eccDNAs. The DNA circularization efficiency afforded by CAES ranged from 15.6% to 31.1%, which was negatively correlated with the eccDNA length. In addition, we demonstrated that CAES-synthesized eccMIRs can express both miRNA-3p and − 5p molecules efficiently independent of a canonical promoter in human cell lines. Further assays proved that these eccMIRs were functional as they were able to repress the luciferase gene containing a miRNA-target sequence in the 3′UTR as well as the endogenous mRNA targets. Finally, kinetics study revealed that eccDNA exhibited a decay rate similar to the standard plasmids and linear DNA in cultured cells. Together, this study offers a rapid and convenient method for Circle-seq users to synthesize artificial eccDNAs. It also demonstrates the promising potential of eccMIR as a bacterial DNA-free vector for safe and robust miRNA overexpression in both basic research and therapeutic applications.

Abstract Background The introduction of adjuvant therapies for patients with resected cutaneous melanoma (CM) has increased the need for sensitive biomarkers for risk stratification and disease monitoring. This study aims to investigate the utility of circulating tumor DNA (ctDNA) assessment in predicting and reflecting disease status during adjuvant therapy. Methods We enrolled 32 patients with resected BRAF-mutated stage III CM receiving adjuvant targeted therapy or immunotherapy. Plasma samples of patients were collected at the baseline (treatment initiation) and during the therapy, and BRAF-mutated ctDNA was quantified by droplet digital PCR (ddPCR). Results Baseline ctDNA was detected in 11/32 (34.4%) patients and predicted postoperative high risk of relapse [HR 3.79, 95% CI 1.20–12.00, p = 0.023]. The three-year overall survival (OS) rate was 54.6% (95% CI 22.9–77.9) versus 95% (95% CI 69.5–99.3) in ctDNA-positive and negative groups, respectively, with significantly worse OS for ctDNA-positive patients [HR 7.92, 95% CI 1.56–40.36, p = 0.013]. Among the baseline ctDNA-positive group (high-risk patients), longitudinal ctDNA detection during adjuvant therapy reflected the clinical outcomes. Only non-relapsing patients cleared their plasma ctDNA by the end of the treatment, while persistent ctDNA detection provided early evidence of disease recurrence. Conclusions ctDNA detection shows promising results in the post-operative setting for identifying cutaneous melanoma patients at the highest risk of relapse and for real-time monitoring of patients’ clinical status and treatment response.

Abstract Background Extracellular vesicles (EVs) are a group of nanoscale cell-derived membranous structures secreted by all cell types, containing molecular cargoes involved in intercellular communication. EVs can be used to mimic “nature’s delivery system” to transport nucleic acids, peptides, lipids, and metabolites to target recipient cells. EVs offer a range of advantages over traditional synthetic carriers, thus paving the way for innovative drug delivery approaches that can be used in different diseases, including cancer. Here, by using breast cancer (BC) cells treated with the multi-kinase inhibitor sorafenib, we generated EVs enriched in specific non-coding RNAs (miR-23b-3p, miR-126-3p, and the long ncRNA GAS5) and investigated their potential impact on the aggressive properties of the BC in vitro and in vivo using zebrafish. Methods EVs were collected from 4 different BC cell lines (HCC1937, MDA-MB-231, MCF-7, and MDA-MB-453) and characterized by western blotting, transmission electron microscopy and nanoparticle tracking analysis. Levels of encapsulated miR-23b-3p, miR-126-3p, and GAS5 were quantified by ddPCR. The role of the EVs as carriers of ncRNAs in vivo was established by injecting MDA-MB-231 and MDA-MB-453 cells into zebrafish embryos followed by EV-based treatment of the xenografts with EVs rich in miR-23b-3p, miR-126-3p and GAS5. Results ddPCR analysis revealed elevated levels of miR-23b-3p, miR-126-3p, and GAS5, encapsulated in the EVs released by the aforementioned cell lines, following sorafenib treatment. The use of EVs as carriers of these specific ncRNAs in the treatment of BC cells resulted in a significant increase in the expression levels of the three ncRNAs along with the inhibition of cellular proliferation in vitro. In vivo experiments demonstrated a remarkable reduction of xenograft tumor area, suppression of angiogenesis, and decreased number of micrometastasis in the tails after administration of EVs enriched with these ncRNAs. Conclusions Our study demonstrated that sorafenib-induced EVs, enriched with specific tumor-suppressor ncRNAs, can effectively inhibit the aggressive BC characteristics in vitro and in vivo. Our findings indicate an alternative way to enrich EVs with specific tumor-suppressor ncRNAs by treating the cells with an anticancer drug and support the development of new potential experimental molecular approaches to target the aggressive properties of cancer cells.

Abstract Detecting EGFR mutations in plasma using droplet digital PCR (ddPCR) assay offers a promising diagnostic tool for lung cancer patients. The performance of plasma-based ddPCR assay relative to traditional EGFR mutation testing in tissue biopsies among Asian patients with suspected lung cancer remains underexplored. Consecutive patients admitted for diagnostic workup for suspected lung cancer were recruited. Peripheral blood samples were collected on the same day of tissue biopsies. Tissue samples were subjected to EGFR mutation analysis via real-time PCR, whereas plasma samples were processed for ddPCR assay to evaluate for EGFR mutation status. The tissue re-biopsy rate was 43.8% while 0.7% of patients failed blood taking. Despite repeat biopsy, 15.2% of patients could not achieve histological diagnosis. Of the 202 patients newly diagnosed with lung cancer, EGFR mutations were detected in 13.4% of plasma samples, compared to 44.3% in tissue samples. Plasma ddPCR for EGFR mutations detection were barely detectable in stages I and II non-small cell lung cancer (NSCLC), but the sensitivity was 25.0%, 56.3%, and 75.0% in stages III, IVA, and IVB NSCLC, respectively. Plasma EGFR mutations were highly specific among all stages of lung cancer. Concordance rates of plasma ddPCR assay also rose with more advanced stages, recorded at 41.9% for stages I and II, 71.9% for stage III, 86.3% for stage IV. In stage IV lung cancer, the false negative rate for the plasma ddPCR assay was 34.4%, whereas that for the tissue testing was 19.2% due to insufficient tissue samples. Plasma-based EGFR genotyping using ddPCR is a non-invasive method that offers early diagnosis and serves as a valuable adjunct to tissue-based testing for patients with advanced-stage lung cancer. However, its usefulness is limited in the context of early-stage lung cancer, indicating a need for further research to improve its accuracy in these patients.

Abstract There is little known about the contribution of exosomal microRNAs (exomiRs) in the children’s cardiac surgery-associated acute kidney injury (CSA-AKI). This study aimed to find diagnostic biomarkers for predicting CSA-AKI in children. A prospective observational study was conducted from April 2020 to March 2021.According to the changes of serum creatinine (SCr) value and urine volume within 48 h, the children were divided into acute kidney injury (AKI) group and non-AKI group. Serum samples were collected 4 h after cardiac surgery. Isolation of extracellular vesicles (EVs) and extraction of exomiRs from serum samples. Illumina high-throughput sequencing was used to quantify exomiRs and screen candidate microRNAs (miRNAs). Expression levels of candidate miRNAs were validated using droplet digital polymerase chain reaction (ddPCR). Normal and injuried rats’ kidney tissue were collected for tissue validation. In the pre-experimental stage (4 AKI vs. 4 non-AKI), hsa-miR-184, hsa-miR-4800-3p, hsa-miR-203a-3p and hsa-miR-6766-3p were selected as candidate genes. In the verification stage (8 AKI vs. 12 non-AKI), the expression of hsa-miR-184 in AKI group was significantly lower than that in non-AKI group (P = 0.031), and the expression of hsa-miR-4800-3p and hsa-miR-6766-3p in AKI group was significantly higher than that in non-AKI group (P = 0.01 and P = 0.047). There was no significant difference in the expression of hsa-miR-203a-3p between the two groups (P > 0.05). The expression of rats’ kidney tissue rno-miR-184 in AKI group was significantly lower than that in the normal group (P = 0.044). The area under the curve (AUC) of AKI predicted by hsa-miR-184 is 0.7865 and the AUC of hsa-miR-6766-3p is 0.7708. Combined with two kinds of miRNAs, the area under the curve of AKI is predicted to be 0.8646. The change of exomiRs level in circulatory system occurred in the early stage after cardiac operation, and the changes of hsa-miR-184 and hsa-miR-6766-3p content in circulatory system could predict CSA-AKI well.

Abstract Background Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples. Results Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R 2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing). Conclusions Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. Video Abstract

Abstract Transient acantholytic dermatosis (TAD) is a relatively common skin disease that typically affects older individuals, which shows clinical and histologic similarities to autosomal dominant Darier disease. TAD was recently shown to be caused by somatic ATP2A2 damaging variants. In this study, we performed Sanger sequencing and droplet digital PCR (ddPCR) in a Japanese elderly male with TAD to identify ATP2A2 somatic mosaicism occurring during embryogenesis for the first time and mutant allelic fraction (MAF). Sanger sequencing revealed a known heterozygous substitution c.1645C>T, p.Arg549* in ATP2A2 from blood and the affected skin containing the epidermis and dermis, whereas the signals of the mutated allele were weaker, suggesting that discrepancy of signal intensities demonstrates the presence of somatic mosaicism. By ddPCR, the normal and mutant allele were identified in genomic DNAs and the MAFs were 20.1% (affected skin) and 14.7% (blood), respectively. In the present case, somatic mosaicisms observed in ectodermal (epidermis) and mesodermal (dermis and blood) origin DNA can be explained by a mutational event during the early‐stage differentiation of embryonic epiblast in embryogenesis, and mutant embryonic cells somewhat preferentially differentiate to form the epidermis, rather than the dermis and blood. Disrupted intracellular Ca2+ homoeostasis through clinical deterioration together with ATP2A2 somatic mosaicism in this patient might result in transient skin eruptions.

Abstract Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

Abstract This study aims to evaluate the prognostic utility of Activity-dependent neuroprotective protein (ADNP) expression in Circulating Tumor Cells (CTCs) inpatients with Non-muscle-invasive Bladder Cancer (NMIBC) undergoing Transurethral Resection of Bladder Tumor (TURBT). A prospective cohort of 74 bladder cancer patients and 22 non-cancer controls were enrolled. The expression of ADNP mRNA was detected by immunomagnetic beads-droplet digital PCR. The ADNP mRNA expression was evaluated in patients with high-risk NMIBC and those with indeterminate invasion depth post 2nd TURBT. Primary cultured bladder cancer cells and PBMCs from healthy donors were immunofluorescence stained. Our findings suggest that baseline ADNP mRNA level in CTCs shows potential as a prognostic marker for NMIBC with a sensitivity of 83.33% and a specificity of 73.58%. In comparison to baseline, ADNP mRNA expression increased post 2nd TURBT in 5 patients, where 2 experienced recurrence. Meanwhile, among the 12 patients with decreased levels, only one patient relapsed. A considerable limitation of this study entails the small sample size. The Immuno-magnetic beads-ddPCR technique provided a viable method for ADNP mRNA detection in CTCs from bladder cancer patients. The preoperative ADNP mRNA level in CTCs was identified as a prognostic indicator for NMIBC. Longitudinal monitoring of ADNP mRNA in CTCs of bladder cancer patients shows promise in evaluating treatment responses and predicting prognosis.

Abstract Malignant melanoma (MM) is known for its abundance of genetic alterations and a tendency for rapid metastasizing. Identification of novel plasma biomarkers may enhance non-invasive diagnostics and disease monitoring. Initially, we examined copy number variations (CNV) in CDK genes (CDKN2A, CDKN2B, CDK4) using MLPA (gDNA) and ddPCR (ctDNA) analysis. Subsequently, low-coverage whole genome sequencing (lcWGS) was used to identify the most common CNV in plasma samples, followed by ddPCR verification of chosen biomarkers. CNV alterations in CDK genes were identified in 33.3% of FFPE samples (Clark IV, V only). Detection of the same genes in MM plasma showed no significance, neither compared to healthy plasmas nor between pre- versus post-surgery plasma. Sequencing data showed the most common CNV occurring in 6q27, 4p16.1, 10p15.3, 10q22.3, 13q34, 18q23, 20q11.21-q13.12 and 22q13.33. CNV in four chosen genes (KIF25, E2F1, DIP2C and TFG) were verified by ddPCR using 2 models of interpretation. Model 1 was concordant with lcWGS results in 54% of samples, for model 2 it was 46%. Although CDK genes have not been proven to be suitable CNV liquid biopsy biomarkers, lcWGS defined the most frequently affected chromosomal regions by CNV. Among chosen genes, DIP2C demonstrated a potential for further analysis.

Introduction Methods Results Conclusion Epigenetics has been shown to be relevant in oncology: BMI1 overexpression has been reported in leukemias, EZH2 mutations have been found in follicular lymphoma, and USP22 seems to stabilize BMI1 protein. In this study, we measured the expression of BMI1, EZH2, and USP22 in lymph nodes from 56 diffuse large B‐cell lymphoma (DLBCL) patients.A new multiplex digital droplet PCR (ddPCR) has been set up to measure the expression of 4 genes (BMI1, EZH2, USP22, and GAPDH) in the same reaction on RNA extracted from paraffin‐embedded tissues.The specificity of ddPCR was confirmed by a 100% alignment on the BLAST platform and its repeatability demonstrated by duplicates. A strict correlation between expression of BMI1 and EZH2 and BMI1 and USP22 has been found, and high expression of these genes was correlated with extra‐nodal lymphomas. Progression‐free survival (PFS) and overall survival (OS) were conditioned by IPI, bone marrow infiltration, and the complete response achievement. High levels of BMI1 and USP22 did not condition the response to therapy, but impaired the PFS, especially for patients defined at “high risk” based on the cell of origin (no germinal center [GCB]), high BCL2 expression, and IPI 3‐5. In this subgroup, the probability of relapse/progression was twice higher than that of patients carrying low BMI1 and USP22 levels.High expression of BMI1 and of USP22 might be a poor prognostic factor in DLBCL, and might represent the target for novel inhibitors. [ABSTRACT FROM AUTHOR]

DNA methylation biomarkers have emerged as promising tools for cancer detection. Common methylation patterns across tumor types allow multi‐cancer detection. Droplet digital PCR (ddPCR) has gained considerable attention for methylation detection. However, multi‐cancer detection using multiple targets in ddPCR has never been performed before. Therefore, we developed a multiplex ddPCR assay for multi‐cancer detection. Based on previous data analyses using The Cancer Genome Atlas (TCGA), we selected differentially methylated targets for eight frequent tumor types (lung, breast, colorectal, prostate, pancreatic, head and neck, liver, and esophageal cancer). Three targets were validated using ddPCR in 103 tumor and 109 normal adjacent fresh frozen samples. Two distinct ddPCR assays were successfully developed. Output data from both assays is combined to obtain a read‐out from the three targets together. Our overall ddPCR assay has a cross‐validated area under the curve (cvAUC) of 0.948. Performance between distinct cancer types varies, with sensitivities ranging from 53.8% to 100% and specificities ranging from 80% to 100%. Compared to previously published single‐target parameters, we show that combining targets can drastically increase sensitivity and specificity, while lowering DNA input. In conclusion, we are the first to report a multi‐cancer methylation ddPCR assay, which allows for highly accurate tumor predictions. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples. Results: Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing). Conclusions: Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. -oGjUmj5e8MXZDxyDjzcm- Video Abstract [ABSTRACT FROM AUTHOR]

Background: Oropouche virus (OROV) is a segmented RNA virus belonging to the genus Orthobunyavirus in the family Peribunyaviridae. Herein, an in-house droplet digital PCR (ddPCR) assay was used for the detection and quantification of OROV. Methods: The ddPCR reaction was assessed as duplex assay using the human housekeeping gene RPP30. Limit of detection (LoD) analysis was performed in whole blood, serum, and urine. The assay was executed on a total of 28 clinical samples (whole blood n = 9, serum n = 11, and urine n = 8), of which 16 specimens were tested positive at the routine molecular diagnostics (endpoint and real-time PCRs). Results: The LoD of the ddPCR performed using 10-fold serial dilution of OROV detected up to 1 cp/µL in all the biological matrices. Compared to the routine molecular diagnostics, the ddPCR assay showed 100% sensitivity for whole blood and serum and 75% for urine, highlighting higher positive rate of ddPCR. Conclusion: We have established a quantitative RNA detection method of OROV with high sensitivity and specificity based on ddPCR. This test is capable of quantitatively monitoring the viral load of OROV and can contribute, in addition to laboratory diagnosis, to shed light on the pathogenesis, filling in the knowledge gaps of this neglected disease and to the vector control programs. [ABSTRACT FROM AUTHOR]

Intraductal papillary mucinous neoplasms (IPMNs) are potential precursor lesions of pancreatic cancer. We assessed the efficacy of screening for KRAS proto‐oncogene, GTPase (KRAS), and GNAS complex locus (GNAS) mutations in cell‐free DNA (cfDNA)—using digital droplet polymerase chain reaction (ddPCR) and circulating epithelial cell (CEC) detection—as biomarkers for risk stratification in IPMN patients. We prospectively collected plasma samples from 25 resected patients at risk of malignant progression, and 23 under clinical surveillance. Our findings revealed KRAS mutations in 10.4% and GNAS mutations in 18.8% of the overall cohort. Among resected IPMN patients, KRAS and GNAS mutation detection rates were 16.0% and 32.0%, respectively, whereas both rates were 4.0% in conservatively managed IPMN. GNAS mutations in cfDNA were significantly more prevalent in resected IPMN (P = 0.024) compared with IPMN under surveillance. No CECs were detected. The absence of KRAS and GNAS mutations could be a reliable marker for branch duct IPMN without worrisome features. The emergence of GNAS mutations could prompt enhanced imaging surveillance. Neither the presence of established worrisome features nor GNAS or KRAS mutations appear effective in identifying high‐grade dysplasia among IPMN patients. [ABSTRACT FROM AUTHOR]

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts. [ABSTRACT FROM AUTHOR]

This study aims to evaluate the prognostic utility of Activity-dependent neuroprotective protein (ADNP) expression in Circulating Tumor Cells (CTCs) inpatients with Non-muscle-invasive Bladder Cancer (NMIBC) undergoing Transurethral Resection of Bladder Tumor (TURBT). A prospective cohort of 74 bladder cancer patients and 22 non-cancer controls were enrolled. The expression of ADNP mRNA was detected by immunomagnetic beads-droplet digital PCR. The ADNP mRNA expression was evaluated in patients with high-risk NMIBC and those with indeterminate invasion depth post 2nd TURBT. Primary cultured bladder cancer cells and PBMCs from healthy donors were immunofluorescence stained. Our findings suggest that baseline ADNP mRNA level in CTCs shows potential as a prognostic marker for NMIBC with a sensitivity of 83.33% and a specificity of 73.58%. In comparison to baseline, ADNP mRNA expression increased post 2nd TURBT in 5 patients, where 2 experienced recurrence. Meanwhile, among the 12 patients with decreased levels, only one patient relapsed. A considerable limitation of this study entails the small sample size. The Immuno-magnetic beads-ddPCR technique provided a viable method for ADNP mRNA detection in CTCs from bladder cancer patients. The preoperative ADNP mRNA level in CTCs was identified as a prognostic indicator for NMIBC. Longitudinal monitoring of ADNP mRNA in CTCs of bladder cancer patients shows promise in evaluating treatment responses and predicting prognosis. [ABSTRACT FROM AUTHOR]

As a famous probiotic, Lacticaseibacillus rhamnosus HN001 is widely added to probiotic products. Different L. rhamnosus strains have different probiotic effects, and the active HN001 strain is the key to exerting probiotic effects, so it is of great practical significance for realising the detection of L. rhamnosus HN001 at the strain level in probiotic products. In this study, strain-specific primer pairs and probes were designed. A combined treatment of sodium deoxycholate (SD) and propidium monoazide (PMA) inhibited the amplification of dead bacterial DNA, establishing a SD-PMA-ddPCR system and conditions for detecting live L. rhamnosus HN001 in probiotic powders. Specificity was confirmed using type strains and commercial strains. Sensitivity tests with spiked samples showed a detection limit of 10⁵ CFU/g and a linear quantification range of 1.42 × 10⁵–1.42 × 10⁹ CFU/g. Actual sample testing demonstrated the method's efficiency in quantifying HN001 in compound probiotic products. This method offers a reliable tool for the rapid and precise quantification of viable L. rhamnosus HN001, crucial for the quality monitoring of probiotic products. [ABSTRACT FROM AUTHOR]

Enterovirus-D68 (EV-D68) is a reemerging virus that has been associated with numerous outbreaks in children in the past 10 years. Most assays examining viral infection kinetics have relied on the use of quantitative RT-PCR (qRT-PCR) assays as an assay of choice. Though valuable, there are inherent limitations that introduce variability, thereby reducing its value when comparing results across the field. Unlike the qRT-PCR assay that uses a standard curve to determine the copy number of viral RNA, the droplet digital PCR assay (ddPCR) directly quantifies the absolute number of copies within a given sample, which in turn makes the assay highly sensitive and accurate. Here, we have developed an EV-D68-specific ddPCR assay that effectively quantifies EV-D68 RNA copies in both cells and supernatants within a dynamic range of 6.7 × 10−3 copies/μL to 1.2 × 104 copies/μL of the sample. The assay was highly specific for a broad range of EV-D68 isolates (Fermon, US/MO/14-18947, US/MO/14-18949, US/KY/14-18953, USA/2018-23088, USA/2020-23336 and EV-D68-infected human nasal turbinate samples from the 2022 outbreak) without cross-reactivity to other viruses such as Enterovirus-A71 (EV-A71), Human Parechovirus (HPeV)-1 and -2, Coxsackievirus (CV)-B1, Human Coronavirus (HCoV)-NL63, SARS-CoV-2, Influenza-A and B, Rhinovirus, and Respiratory Syncytial Virus (RSV)-A2, which are known to cause infection in children. The assay was able to readily quantify EV-D68 in infected cells and supernatants along with nasal turbinate samples collected from children during the 2022 outbreak. Our results suggest that the assay can be readily translated to accurately quantify viral loads in tissues and body fluids such as plasma and lung or nasal aspirates. [ABSTRACT FROM AUTHOR]

Virological evaluation was performed on equine semen to detect the presence of papillomaviruses (PVs) using droplet digital polymerase chain reaction (ddPCR) as the aim of this study was to investigate whether the sperm from asymptomatic stallions harbors ovine papillomaviruses (OaPVs). Twenty-seven semen samples were analyzed, 18 of which were commercially acquired. The remaining nine samples comprising semen and peripheral blood, were collected from nine stallions with no apparent signs of PV-related diseases during clinical examination at the Didactic Veterinary University Hospital (DVUH) of Naples. OaPV was detected in 26 semen samples. OaPV1 was the most prevalent virus infecting equine semen. OaPV1 infected 21 semen samples (~80.8%) and showed a high number of DNA and RNA copies per microliter. qPCR was used to detect OaPV1 DNA in the 18 semen samples. ddPCR was used to detect and quantify the expression of OaPV2, OaPV3, and OaPV4. qPCR failed to detect DNA for these genotypes. Additionally, ddPCR was used to detect the transcriptionally active OaPV1 in six blood and semen samples from the same stallion. ddPCR failed to detect any nucleic acids in OaPVs in peripheral blood samples from the three stallions. In one semen sample, ddPCR detected OaPV1 DNA but failed to detect any nucleic acid in the remaining two semen samples, and peripheral blood from the same animals of the remaining 18 semen samples was not available, OaPV1 and OaPV4 were responsible for nine and five single infections, respectively. No single infections with either OaPV3 or OaPV4 were seen. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae can enter a viable but nonculturable (VBNC) state to survive in unfavorable environments. Our research found that high-, medium-, and low-alcohol-producing K. pneumoniae strains are associated with nonalcoholic fatty liver disease. However, the presence of the three Kpn strains has not been reported in the VBNC state or during resuscitation. In this study, the effects of different strains, salt concentrations, oxygen concentrations, temperatures, and nutrients in K. pneumoniae VBNC state were evaluated. The results showed that high-alcohol-producing K. pneumoniae induced a slower VBNC state than medium-alcohol-producing K. pneumoniae, and low-alcohol-producing K. pneumoniae. A high-salt concentration and microoxygen environment accelerated the loss of culturability. Simultaneously, both real-time quantitative PCR and droplet digital PCR were developed to compare the quantitative comparison of three Kpn strain VBNC states by counting single-copy gene numbers. At 22°C or 37°C, the number of culturable cells decreased significantly from about 108 to 105-106 CFU/mL. In addition, imipenem, ciprofloxacin, polymyxin, and phiW14 inhibited cell resuscitation but could not kill VBNC-state cells. These results revealed that the different environments evaluated play different roles in the VBNC induction process, and new effective strategies for eliminating VBNC-state cells need to be further studied. These findings provide a better understanding of VBNC-state occurrence, maintenance, detection, and absolute quantification, as well as metabolic studies of resuscitation resistance and ethanol production. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Melanoma is the most aggressive skin cancer with ability to recur also after early‐stage tumor surgery. The aim was to identify early‐stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. Methods Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre‐amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. Results The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild‐type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). Conclusion We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. Significance There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early‐stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre‐ and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage.

Abstract This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective.

Precise quantification of the JAK2V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988).

Wastewater surveillance has been a tool for public health officials throughout the COVID-19 pandemic. Universities established pandemic response committees to facilitate safe learning for students, faculty, and staff. These committees met to analyze both wastewater and clinical data to propose mitigation strategies to limit the spread of COVID-19. This paper reviews the initial efforts of utilizing campus data inclusive of wastewater surveillance for SARS-CoV-2 RNA concentrations, clinical case data from university response teams, and mitigation strategies from Grand Valley State University in West Michigan (population 21,648 students) and Oakland University in East Michigan (population 18,552 students) from November 2020 to April 2022. Wastewater positivity rates for both universities ranged from 32.8 to 46.8%. Peak viral signals for both universities directly corresponded to variant points of entry within the campus populations from 2021 to 2022. It was found that the organization of clinical case data and variability of wastewater testing data were large barriers for both universities to effectively understand disease dynamics within the university population. We review the initial efforts of onboarding wastewater surveillance and provide direction for structuring ongoing surveillance workflows and future epidemic response strategies based on those that led to reduced viral signals in campus wastewater. HIGHLIGHTS Viral RNA levels in wastewater tracked the emergence of variants in student populations.; Intervention strategies suggested reduced numbers in wastewater viral RNA signals prior to variant emergence.; Public health promotion and educational tools are needed to take complex biological processing to actionable intervention strategies.;

Abstract Background Gastrointestinal nematodes (GINs) have a major impact on sheep production, health, and welfare worldwide. Norway is no exception, but there are only a few studies on the prevalence of GINs in Norwegian sheep. The aim of this study was to investigate the current occurrence of the most important nematodes in sheep flocks in Norway. Faecal samples were collected from flocks in 2021/2022, mainly from three geographical regions in Norway, i.e., northern, eastern, and western. In each of 134 flocks included, individual samples from 10 lambs (autumn) were pooled. Third stage larvae (L3) were cultivated and harvested (Baermann method) from the pooled samples. The DNA was then extracted and further analysed using droplet digital PCR (ddPCR). This enables assessment of the proportions of the three most important nematode species/genera, i.e., H. contortus, T. circumcincta, and Trichostrongylus. The fractional abundance/relative proportion of each species/genus was assessed by performing duplex assays with universal strongyle and species/genus-specific primers and probe sets. In addition, the occurrence of Nematodirus eggs was assessed by standard faecal egg counts (i.e., McMaster method). Results Of the 134 flocks sampled, 24 were from the northern region, 31 from eastern, and 71 from western Norway. In addition, some flocks from central (n = 7), and southern (n = 1) Norway were included. Among the sampled flocks, T. circumcincta occurred most commonly (94%), followed by H. contortus (60%) and Trichostrongylus (55%), and Nematodirus (51%). In general, mixed infections were observed, with 38% and 18% of flocks infected with three or all four genera, respectively. Conclusions The results of this study indicate that GINs are widespread in Norway. Teladorsagia circumcincta seems to be present in most flocks based on this screening. Moreover, the results show that Nematodirus spp. infect lambs throughout the country, predominantly N. battus, and indicate that this nematode has become more abundant, which could lead to an increase in nematodirosis.

Abstract Background Droplet digital PCR (ddPCR) is widely applied to monitor measurable residual disease (MRD). However, there are limited studies on the feasibility of ddPCR-MRD monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially targeting multiple molecular markers simultaneously. Methods Our study collected samples from patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) in complete remission after allo-HSCT between January 2018 and August 2021 to evaluate whether posttransplant ddPCR-MRD monitoring can identify patients at high risk of relapse. Results Of 152 patients, 58 (38.2%) were MRD positive by ddPCR within 4 months posttransplant, with a median variant allele frequency of 0.198%. The detectable DTA mutations (DNMT3A, TET2, and ASXL1 mutations) after allo-HSCT were not associated with an increased risk of relapse. After excluding DTA mutations, patients with ddPCR-MRD positivity had a significantly higher cumulative incidence of relapse (CIR, 38.7% vs. 9.7%, P

DNA methylation is characterized as a gene regulatory mechanism that involves the methylation of the 5-carbon (C5) position of cytosine, resulting in the formation of 5-methylcytosine. The analysis of aberrantly methylated cytosine-phosphate-guanine (CpG) dinucleotides, primarily in the promoter regions of tumor suppressor genes, can serve as promising prognostic and predictive markers of cancer development. Meis homeobox 1 (MEIS1) gene, crucial for cell growth and differentiation, exhibits dysregulation linked to various cancer types, acting as both a positive and negative regulator. The selection of an appropriate method for the evaluation of gene promoter methylation status is important for clinical implementation without biases regarding false positive and false negative outcomes. The study focuses on the optimization of a novel droplet digital PCR (ddPCR) assay for identifying the methylation status of MEIS1. Compared to traditional methods, ddPCR offers an increased sensitivity and specificity, presenting a promising tool for precise DNA methylation assessment with potential implications for cancer diagnostics and prognostics.

FNDC3B (fibronectin type III domain containing 3B) is highly expressed in hepatocellular carcinoma (HCC) and other cancer types, and fusion genes involving FNDC3B have been identified in HCC and leukemia. Growing evidence suggests the significance of FNDC3B in tumorigenesis, particularly in cell migration and tumor metastasis. However, its regulatory mechanisms remain elusive. In this study, we employed bioinformatic, gene regulation, and protein‐DNA interaction screening to investigate the transcription factors (TFs) involved in regulating FNDC3B. Initially, 338 candidate TFs were selected based on previous chromatin immunoprecipitation (ChIP)‐seq experiments available in public domain databases. Through TF knockdown screening and ChIP coupled with Droplet Digital PCR assays, we identified that E2F1 (E2F transcription factor 1) is crucial for the activation of FNDC3B. Overexpression or knockdown of E2F1 significantly impacts the expression of FNDC3B. In conclusion, our study elucidated the mechanistic link between FNDC3B and E2F1. These findings contribute to a better understanding of FNDC3B in tumorigenesis and provide insights into potential therapeutic targets for cancer treatment.

This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective. [ABSTRACT FROM AUTHOR]

Viruses from Picornaviridae family are known pathogens of poultry, although the information on their occurrence and pathogenicity in pigeons is scarce. In this research, efforts are made to broaden the knowledge on Megrivirus B and Pigeon picornavirus B prevalence, phylogenetic relationship with other avian picornaviruses and their possible connection with enteric disease in racing pigeons. As a result of Oxford Nanopore Sequencing, five Megrivirus and two pigeon picornavirus B-like genome sequences were recovered, among which three recombinant strains were detected. The recombinant fragments represented an average of 10.9% and 25.5% of the genome length of the Pigeon picornavirus B and Megrivirus B reference strains, respectively. The phylogenetic analysis revealed that pigeons are carriers of species-specific picornaviruses. TaqMan qPCR assays revealed 7.8% and 19.0% prevalence of Megrivirus B and 32.2% and 39.7% prevalence of Pigeon picornavirus B in the group of pigeons exhibiting signs of enteropathy and in the group of asymptomatic pigeons, respectively. In turn, digital droplet PCR showed a considerably higher number of genome copies of both viruses in sick than in asymptomatic pigeons. The results of quantitative analysis leave the role of picornaviruses in enteropathies of pigeons unclear. [ABSTRACT FROM AUTHOR]

Although several promising approaches for the treatment of relapsed/refractory diffuse large B‐cell lymphoma (rrDLBCL) have been approved recently, it remains unclear which patients will ultimately achieve long‐term responses. Circulating tumor (ct)DNA sequencing has emerged as a valuable tool to assess minimal residual disease (MRD). Correlations between MRD and outcomes have been shown in previously untreated DLBCL, but data on the repeated assessment of MRD in the dynamic course of rrDLBCL is limited. Here, we present an approach leveraging cost‐ and time‐sensitivity of digital droplet (dd)PCR to repeatedly assess MRD in rrDLBCL and present proof‐of‐principle for its ability to predict outcomes. [ABSTRACT FROM AUTHOR]

Precise quantification of the JAK2V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations. [ABSTRACT FROM AUTHOR]