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IntroductionAltered immune signatures are emerging as a central theme in neurodegenerative disease, yet little is known about immune responses in early-onset Alzheimer's disease (EOAD).MethodsWe examined single-cell RNA-sequencing (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) and droplet digital polymerase chain reaction (ddPCR) data from CD4 T cells from participants with EOAD and clinically normal controls.ResultsWe analyzed PBMCs from 16 individuals by scRNA-seq and discovered increased interferon signaling-associated gene (ISAG) expression and striking expansion of antiviral-like ISAGhi T cells in EOAD. Isolating CD4 T cells from 19 individuals, including four cases analyzed by scRNA-seq, we confirmed increased expression of ISAGhi marker genes. Publicly available cerebrospinal fluid leukocyte scRNA-seq data from late-onset mild cognitive impairment and AD also revealed increased expression of interferon-response genes.DiscussionAntiviral-like ISAGhi T cells are expanded in EOAD. Additional research into these cells and the role of heightened peripheral IFN signaling in neurodegeneration is warranted.HighlightsInterferon-responsive T cells expanded in early-onset Alzheimer's disease (AD). Increased interferon-associated gene expression present in early- and late-onset AD. Peripheral immune changes in T and NK cells driven by females with early-onset AD.

Background: Droplet digital PCR (ddPCR) is a highly sensitive tool for detecting bacterial DNA in bacterial bloodstream infections (BSI). This study aimed to examine the sensitivity and specificity of ddPCR and the association between bacterial DNA load in whole blood and the time-to-positivity (TTP) of blood culture (BC) in patients with Escherichia coli BSI. Methods: This prospective study enrolled patients with E. coli BSI confirmed via BC at the Hiroshima University Hospital from June 2023 to August 2024. The E. coli DNA load in whole blood, which was simultaneously obtained from two BC sets, was measured using ddPCR with E. coli specific primer and probe. Whole blood samples from 50 patients with BC positive for pathogens other than E. coli (n = 25) and BC negative (n = 25) were also evaluated using ddPCR. Results: A total of 131 patient samples were analyzed in this study. Of the 81 patients with E. coli BSI, ddPCR detected E. coli DNA in 67 (82.7%). The results of ddPCR for E. coli had a sensitivity of 82.7% (95% CI: 73.1–89.4%), specificity 100% (95% CI: 93.0–100%). Patients with positive ddPCR results had significantly shorter TTP than those with negative results (median, 8.8 h vs. 10.7 h, p < 0.001). The positivity rate for both BC sets was significantly higher in patients with positive ddPCR results than in those with negative results (89.6% vs. 35.1%, p < 0.001). Among ddPCR-positive patients, septic shock was significantly associated with intestinal perforation, higher E. coli DNA load, higher 28-d mortality, shorter TTP, and higher positivity rate for four bottles of BC than those without septic shock. The E. coli DNA load in whole blood negatively correlated with TTP (p < 0.001, R2 = 0.38). Conclusion: The E. coli DNA load in whole blood is inversely correlated with TTP. Notably, a higher E. coli DNA load is associated with septic shock. [ABSTRACT FROM AUTHOR]

Plasma cell‐free DNA (cfDNA) analysis to track estrogen receptor 1 (ESR1) mutations is highly beneficial for the identification of tumor molecular dynamics and the improvement of personalized treatments for patients with metastatic breast cancer (MBC). Plasma‐cfDNA is, up to now, the most frequent liquid biopsy analyte used to evaluate ESR1 mutational status. Circulating tumor cell (CTC) enumeration and molecular characterization analysis provides important clinical information in patients with MBC. In this study, we investigated whether analysis of CTCs and circulating tumor DNA (ctDNA) provide similar or complementary information for the analysis of ESR1 mutations. We analyzed both plasma‐cfDNA (n = 90) and paired CTC‐derived genomic DNA (gDNA; n = 42) from 90 MBC patients for seven ESR1 mutations. Eight out of 90 (8.9%) plasma‐cfDNA samples tested using the ddPLEX Mutation Detection Assay (Bio‐Rad, Hercules, CA, USA), were found positive for one ESR1 mutation, whereas 11/42 (26.2%) CTC‐derived gDNA samples were found positive for at least one ESR1 mutation. Direct comparison of paired samples (n = 42) revealed that the ESR1 mutation rate was higher in CTC‐derived gDNA (11/42, 26.2%) than in plasma‐cfDNA (6/42, 14.3%) samples. Our results, using this highly sensitive ddPLEX assay, reveal a higher percentage of mutations in CTC‐derived gDNAs than in paired ctDNA in patients with MBC. CTC‐derived gDNA analysis should be further evaluated as an important and complementary tool to ctDNA for identifying patients with ESR1 mutations and for guiding individualized therapy. [ABSTRACT FROM AUTHOR]

Gliomas are the most common brain tumor type in children and adolescents. To date, diagnosis and therapy monitoring for these tumors rely on magnetic resonance imaging (MRI) and histopathological as well as molecular analyses of tumor tissue. Recently, liquid biopsies (LB) have emerged as promising tool for diagnosis and longitudinal tumor assessment potentially allowing for a more precise therapeutic management. However, the optimal strategy for monitoring gliomas by LB remains to be determined. In this study, we analyzed circulating tumor DNA (ctDNA) from 78 liquid biopsies (plasma n = 44, cerebrospinal fluid n = 34 (CSF)) of 35 glioma patients, determining H3F3A K28M (K27M) and BRAF V600E mutation allele frequency using droplet digital PCR (ddPCR). All results were correlated to clinically relevant parameters including diagnostic imaging and CSF aspiration site (ventricular vs lumbar) with respect to tumor localization. Regarding diagnostic accuracy, the calculated sensitivity score in the H3F3A K27M cohort was 84.61% for CSF and 73.68% for plasma. In the BRAF V600E cohort, we determined a sensitivity of 83.3% in plasma and 80% in CSF. The overall specificity was 100%. With respect to the CSF aspiration, the intra-operatively obtained CSF demonstrated 100% detection rate, followed by ventricular CSF obtained via Ommaya Reservoir/shunt puncture (93%) and CSF obtained via lumbar puncture (66%). Notably, this further correlated with the proximity of the CSF site to tumor localization. Longitudinal CSF monitoring demonstrated a good correlation to clinical and radiological disease evolution. Importantly, we show for the first time that monitoring BRAF V600E by ddPCR could serve as treatment response assessment in gliomas. In summary, our observation may inform recommendations with regard to location of CSF aspiration when incorporating LB into future treatment protocols. [ABSTRACT FROM AUTHOR]

Animal‐related crimes have increased with an increase in the number of pets worldwide, underscoring the importance of animal‐related biological evidence at crime scenes. Evidence obtained in cases involving dogs and cats often includes a mixture of human and animal DNA. In this study, we developed a method using droplet digital polymerase chain reaction (ddPCR) to simultaneously identify and quantitatively detect human, dog, and cat DNA in mixed samples. HLA‐DRA was chosen as a human‐specific marker, OR6D7 as a dog‐specific marker, and FLAI‐K as a cat‐specific marker. The species specificity of each target was confirmed using 14 control DNA samples from 11 mammals and 3 birds. Sensitivity tests determined the limit of detection to be 0.0008 ng/μL for human DNA and 0.00061 ng/μL for dog and cat DNA. In the mixture test, each DNA sample was independently and accurately detected in samples containing trace amounts of all three types of DNA. This study demonstrated the effectiveness of applying ddPCR to forensic case samples from dog‐ and cat‐related incidents. We have presented a reliable method for the accurate identification and quantification of human, dog, and cat DNA simultaneously, offering possibilities for advancements in forensic DNA analysis and related fields. [ABSTRACT FROM AUTHOR]

Introduction: Detection of PIK3CA mutations in primary tumors and liquid biopsy samples is of increasing importance for treatment decisions and therapy resistance in many types of cancer. The aim of the present study was to directly compare the efficacy of a relatively inexpensive ultrasensitive real-time PCR with the well-established and highly sensitive technology of ddPCR for the detection of the three most common hotspot mutations of PIK3CA , in exons 9 and 20, that are all of clinical importance in various types of cancer. Patients and methods: We analyzed 42 gDNAs from primary tumors (FFPEs), 29 plasma-cfDNA samples, and 29 paired CTC-derived gDNAs, all from patients with ER+ metastatic breast cancer, and plasma from 10 healthy donors. The same blood draws were used for CTC isolation using EpCAM beads for positive immunomagnetic enrichment. All FFPEs and plasma-cfDNA samples were analyzed in parallel for PIK3CA mutations by ultrasensitive real-time PCR assay and droplet digital PCR. Results: In gDNAs from FFPEs, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 22/42(52.4%), and the p.E542K and p.H1047R mutations were detected in 14/42(33.3%) and 16/42(38.1%), respectively. Using ddPCR, the p.E545K mutation was detected in 22/42(52.4%), p.E542K in 17/42(40.5%), and p.H1047R in 19/42(45.2%) samples, revealing a concordance between the two methodologies of 81%, 78.6% and 78.6% for each mutation respectively. In plasma-cfDNA, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 11/29(38%) and both p.E542K and p.H1047R mutations in 2/29(6.9%).In the same plasma-cfDNA samples using ddPCR, p.E545K was detected in 1/29(3.5%), p.E542K in 2/29(6.9%), and p.H1047R in 3/29(10.5%) samples, revealing a concordance of 65.5%,100% and 93.1% for each mutation respectively. In paired CTC-derived gDNAs p.E545K was detected in 11/29(38%), p.E542K in 3/29(10.3%), and p.H1047R in 7/29(24.1%) samples. Conclusions: This low-cost, high-throughput and ultrasensitive real-time PCR assay provides accurate and specific detection of PIK3CA hotspot mutations in liquid biopsy samples and gives similar results to ddPCR. This assay can be performed in labs where digital PCR instrumentation is not available. In CTC-derived gDNA and paired plasma-cfDNA, PIK3CA mutations detected were not identical, revealing that CTC and plasma-cfDNA give complementary information. [ABSTRACT FROM AUTHOR]

Transient abnormal myelopoiesis (TAM) generally affects newborns with Down syndrome and is associated with constitutional trisomy 21 and a somatic GATA1 mutation. Here we describe a case of TAM which evolved after umbilical cord blood transplantation (UCBT), whose origin was identified as a GATA1 mutation-harboring clone in umbilical cord blood (UCB) by detailed genetic analyses. A 58-year-old male who received UCBT for peripheral T-cell lymphoma presented progressive anemia and thrombocytopenia, and leukocytosis with blast cells in the peripheral blood (PB). Bone marrow (BM) aspiration showed granulocytic and megakaryocytic dysplasia with excess blasts whose karyotype was trisomy 21. Short tandem repeat analysis showed complete donor chimerism. He was initially diagnosed as donor-derived myelodysplastic syndrome (MDS) and treated with azacitidine, followed by secondary transplantation using unrelated BM, providing durable complete remission. Retrospective targeted-capture sequencing analysis of PB/BM samples collected at multiple timepoints identified trisomy 21 and a GATA1 mutation, suggestive of a diagnosis of donor cell-derived TAM (DC-TAM). Importantly, a minor clone with the same GATA1 mutation was detected in UCB by droplet digital PCR. DC-TAM is a rare UCBT-related complication which resembles MDS, but the identification of GATA1 mutation may be useful for its diagnosis. Our genetic analyses revealed that a pre-existing clone in UCB may contribute to the development of donor cell-derived hematologic neoplasms, highlighting the potential relevance of genetic screening of donor UCB. [ABSTRACT FROM AUTHOR]

This paper reported a novel approach to quantification of adulterated fox-derived components in meat products by drop digital polymerase chain reaction (ddPCR). By using the F2 gene as the target gene of fox, a single primer was designed to identify the adulteration that had been added either inadvertently or deliberately during the process. In this paper, the fox meat was used as the experimental materials and a relationship was established between fox mass and copy number by extracting DNA and using DNA concentration as an intermediary. The results that across the dynamic range, the relationships between meat mass and DNA concentration were nearly linear (R2 = 0.9986), as was the relationship between DNA concentration and DNA copy number (R2 = 0.9992). Based on the DNA concentration, the following formulas were developed about the relationship between fox meat mass (Mfox) and DNA copy number (C): Mfox = 0.05C + 2.7. [ABSTRACT FROM AUTHOR]

Chinese rice‐field eels rhabdovirus (CrERV) causes haemorrhagic disease in Chinese rice‐field eels (Monopterus albus), leading to significant mortality and economic losses. Sensitive detection of CrERV nucleic acids is essential to control the spread of this pathogen and to treat infected individuals. Herein, we developed an efficient and sensitive droplet digital PCR (ddPCR) method to rapidly detect and quantify CrERV. The ddPCR assay optimal conditions were an annealing temperature of 53°C, and primer and probe concentrations of 0.5 and 0.25 μM, respectively. The assay had a diagnostic sensitivity of 0.23 copies/μL, and was highly specific, showing no cross reactivity with other viruses (infectious haematopoietic necrosis virus, grass carp reovirus, spring viremia of carp virus, largemouth bass ranavirus, carp edema virus, Chinese giant salamander iridovirus, and white spot syndrome virus). Real‐time quantitative PCR testing of 30 Chinese rice‐field eels samples detected CrERV in 7 samples (23.3%), whereas ddPCR detected CrERV in 12 samples (40%), demonstrating its higher sensitivity. Thus, ddPCR represents an advanced method to absolutely quantify CrERV in infected fish with low virus concentrations, providing a valuable tool to manage the spread and impact of CrERV. [ABSTRACT FROM AUTHOR]

This study provides a comprehensive summary of the findings regarding the application and diagnostic efficacy of droplet digital PCR (ddPCR) in detecting viral and bacterial pathogens in aquaculture. Utilizing a systematic search of four databases up to 6 November 2023, we identified studies where ddPCR was deployed for pathogen detection in aquaculture settings, adhering to Preferred Reporting Items for Systematic Reviews and Meta‐analysis of Diagnostic Test Accuracy guidelines. From the collected data, 16 studies retrieved, seven were included in a meta‐analysis, encompassing 1121 biological samples from various fish species. The detection limits reported ranged markedly from 0.07 to 34 copies/μL. A direct comparison of the diagnostic performance between ddPCR with quantitative PCR (qPCR) proved challenging due to limited data, thus only a pooled sensitivity analysis was feasible. The results showed a pooled sensitivity of 0.750 (95% confidence interval [CI]: 0.487–0.944) for ddPCR, compared to 0.461 (95% CI: 0.294–0.632) for qPCR, with no statistically significant difference in sensitivity between the two methods (p =.5884). Notably, significant heterogeneity was observed among the studies (I2 = 93%–97%, p <.01), with the year of publication significantly influencing this heterogeneity (p <.001), but not the country of origin (p =.49). No publication bias was detected, and the studies generally exhibited a low risk of bias according to QUADAS‐C criteria. While ddPCR and qPCR showed comparable sensitivities in pathogen detection, ddPCR's capability to precisely quantify pathogens without the need for standard curves highlights its potential utility. This characteristic could significantly enhance the accuracy and reliability of pathogen detection in aquaculture. [ABSTRACT FROM AUTHOR]

Background: Sporadic desmoid fibromatosis (DF) is a rare locally aggressive tumor characterized by mutation in exon 3 of CTNNB1 (T41A, S45F, and S45P). Standard of care is active surveillance (AS), but 30% require treatment. DF clinical course is unpredictable and identification of prognostic markers is needed to tailor strategy. In this prospective study, we investigated the consistency between mutation detected in tumor biopsies with that detected in plasma by digital droplet PCR (ddPCR) and the association between circulating tumor DNA (ctDNA) abundancy with clinical outcome. Patients and Methods: A total of 56 patients and 10 healthy donors were included. CTNNB1 mutation status of DF biopsies was determined by Sanger and in case of WT CTNNB1 with NGS. In matched plasma samples at enrollment and during AS at specific timepoints, we evaluated cfDNA quantity and ctDNA. Results: ctDNA levels were measured in 46 patients with CTNNB1 mutation. Detection rate for T41A, S45F and S45P was 68%, 42% and 100%, respectively. S45P variant has been detected in all patients with S45P mutation. Longitudinal assessment of ctDNA during AS in nine patients (four with regression and five with progression as first event according to RECIST) showed a concordance between the event and ctDNA level change in six out of nine patients tested (4/5 with progression and 2/4 with regression). Conclusions: Results of ctDNA analysis support its potential clinical implementation as diagnostic tool in specific clinical scenarios where biopsy can be challenging. A prospective clinical trial needs to be performed to evaluate the potential role of ctDNA as predictive biomarker. [ABSTRACT FROM AUTHOR]

Rotaviruses are well‐recognized pathogens responsible for diarrhea in humans and various animal species, with Rotavirus A the most often detected and most thoroughly described. Rotaviral disease is an important concern in pathology of pigeons as well, as pigeon rotavirus A was proven to play a major role in young pigeon disease (YPD). However, rotaviruses of other groups have been so far understudied in birds. This paper describes the first finding of Rotavirus G in domestic pigeon in Europe, as well as the recovery of three complete genomes of pigeon rotavirus A with Oxford Nanopore Sequencing. Quantification of pigeon rotavirus A genetic material with droplet digital polymerase chain reaction (PCR) in pigeons suffering from diarrhea and in asymptomatic pigeons was also performed in the frameworks of this study and resulted in determination of statistically highly significant differences between the groups in both detection rate and shedding of the virus. Phylogenetic analysis revealed the close relationship of acquired strains with those originating from pigeons from Europe, North America, Asia, and Australia, indicating a broad geographical spread of pigeon rotaviruses. Results of our research shed more light on occurrence and diversity of Rotavirus species in pigeons. [ABSTRACT FROM AUTHOR]

Background: The 1986 disaster at the Chornobyl Nuclear Power Plant released massive amounts of radioactive material into the local environment. In addition to radiation, remediation efforts and abandonment of military-industrial complexes contributed to contamination with heavy metals, organics, pesticides and other toxic chemicals. Numerous studies have evaluated the effects of this contamination on the local ecology. However, few studies have reported the effect of this contamination on vector-borne pathogens and their hosts. In this manuscript, we characterize tick-borne pathogen presence at two sample locations within the Chornobyl Exclusion Zone, one at the Nuclear Power Plant (NPP) and another 16 km away in Chornobyl City (CC). Methods: Ticks and whole-blood samples were collected from free-breeding dogs captured at the NPP and CC. Endpoint PCR and quantitative PCR were used to identify tick species and to assess the presence of specific tick-borne pathogens, including Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Babesia spp., Bartonella spp., Francisella tularensis and general Anaplasmataceae. A droplet digital PCR assay was developed for Babesia canis and A. phagocytophilum to evaluate their presence in dogs from the two populations. Pathogen prevalences between the two sample populations were compared by calculating Z-scores. Results: Ticks were identified as Ixodes ricinus (n = 102) and Dermacentor reticulatus (n = 4). Overall, 56.9% of I. ricinus ticks were positive for at least one pathogen. A significantly higher prevalence of A. phagocytophilum and B. burgdorferi was found in ticks at the NPP (44.0% and 42.0%, respectively) compared to CC (23.1% and 19.2%, respectively). Babesia spp. (including B. canis and B. caballi) were detected in 8.8% ticks at similar proportions for both populations. Interestingly, we found a significantly lower level of A. phagocytophilum in dogs at the NPP (1.8%) than in dogs at CC (11.7%). In total, 24.3% of dogs were positive for B. canis, evenly distributed across the two populations. Conclusions: The results of this study show contrasting pathogen prevalence in both ticks and dogs at the NPP and CC, which may reflect the differential exposures at the two locations. This work adds an important new component to our understanding of the consequences of prolonged exposure to environmental contamination on the wildlife and ecology within the Chornobyl Exclusion Zone. [ABSTRACT FROM AUTHOR]

In order to realize high efficiency and high precision quarantine identification and epidemic monitoring of Pseudomonas savastanoi pv. phaseolicola (Psp),the detection method of Psp by droplet digital PCR (ddPCR) was established. The ddPCR reaction system was conducted with Psp as research object and site-specific recombinases were used as gene target with SSRP-F/ SSRP-R/ SSRP-P as primers and probe. The results showed that the detection limit of genomic DNA by ddPCR was 0. 6 copies/ μL. The detection sensitivity of ddPCR could increase two orders of magnitude than convention PCR. Meanwhile, the activity of Psp carried in peas was studied, which improved the epidemic detection rate. [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) poses a substantial threat to diverse carnivorans, leading to systemic and often fatal diseases. Accurate and prompt diagnosis is paramount for effective management and curbing further transmission. This study evaluates the diagnostic performance of droplet digital PCR (ddPCR) in comparison to conventional reverse-transcription (RT-PCR) and quantitative reverse-transcription real-time PCR (RT-qPCR). Seventy-six clinical samples were collected from dogs with CDV symptoms diagnosed by specialized veterinarians, and sixteen samples from apparently healthy individuals. Conventional PCR, quantitative real-time PCR, and ddPCR were deployed, and their diagnostic capabilities were meticulously assessed. DdPCR exhibited heightened analytical sensitivity, reaching a detection limit of 3 copies/μL, whereas RT-qPCR had a detection limit of 86 copies/μL. The comparative analysis between clinical diagnosis and molecular techniques, including RT-PCR and RT-qPCR, demonstrated low concordance, with Kappa coefficients of 0.268 and 0.324, respectively. In contrast, ddPCR showed a moderate concordance, with a Kappa coefficient of 0.477. The sensitivity was 42.4% for RT-PCR, 57.9% for RT-qPCR, and 72.4% for ddPCR, with 100% specificity for all methods. This study underscores ddPCR's superior sensitivity and agreement with clinical CDV diagnosis, even at low viral concentrations, suggesting it as a promising alternative for CDV diagnosis. [ABSTRACT FROM AUTHOR]

With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy. [ABSTRACT FROM AUTHOR]

In this study, a ddPCR method for the detection of scale drop disease virus (SDDV) in yellowfin seabream (Acanthopagrus latus) was established based on Real-time fluorescence quantitative PCR detection methods and principles. The reaction conditions were optimized, and the sensitivity, specificity, accuracy, and reproducibility were assessed. The results showed that threshold line position was determined to be 1900 by the ddPCR method; the optimum annealing temperature for SDDV detection by the ddPCR method was 60°C; the limit of detection was 1.4-1.7 copies/µL; the results of specific detection of other common viruses, except for SDDV specific amplification, were all negative; and the relative standard deviation (RSD) for the reproducibility validation was 0.77%. The samples of yellowfin seabream (Acanthopagrus latus) liver, spleen, kidney, heart, intestine, brain, blood, muscle, skin and ascites with three replicates, respectively, were tested using the ddPCR method, and the results were consistent with clinical findings. The ddPCR method established in this study has the advantages of high sensitivity, high specificity, good reproducibility and simple steps for the quantitative detection of SDDV, which could be used for the nucleic acid detection of clinical SDDV samples, and provided a new quantitative method for the diagnosis of yellowfin seabream SDDV in the early stage of pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Droplet digital (dd)PCR is a new-generation PCR technique with high precision and sensitivity; however, the positive and negative droplets are not always effectively separated because of the “rain” phenomenon. We aimed to develop a practical optimization and evaluation process for the ddPCR assay and to apply it to the detection of BRAF V600E in fine-needle aspiration (FNA) specimens of thyroid nodules, as an example. Methods: We optimized seven ddPCR parameters that can affect “rain.” Analytical and clinical performance were analyzed based on histological diagnosis after thyroidectomy using a consecutive prospective series of 242 FNA specimens. Results: The annealing time and temperature, number of PCR cycles, and primer and probe concentrations were found to be more important considerations for assay optimization than the denaturation time and ramp rate. The limit of blank and 95% limit of detection were 0% and 0.027%, respectively. The sensitivity of ddPCR for histological papillary thyroid carcinoma (PTC) was 82.4% (95% confidence interval [CI], 73.6%–89.2%). The pooled sensitivity of BRAF V600E in FNA specimens for histological PTC was 78.6% (95% CI, 75.9%–81.2%, I² =60.6%). Conclusions: We present a practical approach for optimizing ddPCR parameters that affect the separation of positive and negative droplets to reduce rain. Our approach to optimizing ddPCR parameters can be expanded to general ddPCR assays for specific mutations in clinical laboratories. The highly sensitive ddPCR can compensate for uncertainty in cytological diagnosis by detecting low levels of BRAF V600E. [ABSTRACT FROM AUTHOR]

Abstract Background Droplet digital PCR (ddPCR) is a highly sensitive tool for detecting bacterial DNA in bacterial bloodstream infections (BSI). This study aimed to examine the sensitivity and specificity of ddPCR and the association between bacterial DNA load in whole blood and the time-to-positivity (TTP) of blood culture (BC) in patients with Escherichia coli BSI. Methods This prospective study enrolled patients with E. coli BSI confirmed via BC at the Hiroshima University Hospital from June 2023 to August 2024. The E. coli DNA load in whole blood, which was simultaneously obtained from two BC sets, was measured using ddPCR with E. coli specific primer and probe. Whole blood samples from 50 patients with BC positive for pathogens other than E. coli (n = 25) and BC negative (n = 25) were also evaluated using ddPCR. Results A total of 131 patient samples were analyzed in this study. Of the 81 patients with E. coli BSI, ddPCR detected E. coli DNA in 67 (82.7%). The results of ddPCR for E. coli had a sensitivity of 82.7% (95% CI: 73.1–89.4%), specificity 100% (95% CI: 93.0–100%). Patients with positive ddPCR results had significantly shorter TTP than those with negative results (median, 8.8 h vs. 10.7 h, p

Barley yellow dwarf viruses (BYDVs) belong to a complex of several species, all vectored by aphids. Due to the abundance of Sitobion avenae and Rhopalosiphum padi, BYDV-MAV and BYDV-PAS are among the prevalent species in Irish crops. Several BYDV detection methods, such as immunosorbent assays and PCR-based diagnostic tests, are available and routinely used. However, there are opportunities to develop improved assays to capture viral load information from different sample matrices. Here, we successfully developed a droplet digital PCR assay to detect and quantify BYDV-MAV and BYDV-PAS in both aphid and barley samples. The high specificity shown by this assay allows us to differentiate the two species from each other within a wide dynamic range. This assay will provide a better overview of the process underlying BYDV infection and transmission from the early stage of infection to the appearance of the symptoms.

Abstract Background Acne vulgaris is a multifactorial disease of the pilosebaceous unit of human skin. Previous studies have identified an acne‐associated dysbiosis of the skin microbiome. Objectives This dysbiosis was mainly determined for Cutibacterium acnes. However, detailed analyses combining qualitative and quantitative aspects are scarce, also regarding the possible contribution of other skin bacteria and the impact of treatment. Methods We conducted a culture‐independent study to determine differences between the healthy skin and the acne microbiome before and after isotretinoin treatment. Three amplicon‐based sequencing approaches and digital droplet PCR for quantification were applied. Results Our results revealed a 2.2‐fold reduced abundance of C. acnes with a reduced diversity in the acne microbiome. A phylotype switch was found, which was mainly characterized by a significant relative decrease of IB and II strains in the acne microbiome. In contrast, the relative abundance of staphylococci increased significantly and the quantitative ratio of staphylococci to C. acnes strongly increased from 1:34 in the healthy cohort to 1:11 in the acne cohort. The diversity of staphylococci was reduced, mainly due to the decrease of Staphylococcus hominis, and the appearance and predominance of Staphylococcus aureus in some acne patients. Isotretinoin treatment drastically depleted C. acnes (37‐fold) and moderately also staphylococci (3.6‐fold). Isotretinoin treatment resulted in a decrease of Staphylococcus epidermidis and a significant increase of S. aureus on facial skin. Conclusions The switch from a C. acnes‐dominated healthy skin microbiome towards an acne microbiome that is relatively enriched in staphylococci could indicate a stronger impact of staphylococci in the pathophysiology of acne than currently acknowledged. Our data further showed that isotretinoin largely eliminated the skin microbiome and in particular C. acnes, but also S. epidermidis. Instead, more harmful bacteria such as S. aureus could expand, suggesting that posttreatment strategies should be considered to accelerate skin microbiome recovery.

Abstract Domestic dogs (Canis lupus familiaris) serve as valuable translational models for human cancer research due to their biological similarities. Canine mammary tumors (CMTs), frequently diagnosed in female dogs, share various characteristics with human breast cancers. This study investigates the PIK3CA (H1047R) mutation in CMTs using droplet digital PCR (ddPCR) and explores the potential of liquid biopsy for non-invasive detection. We analyzed 80 formalin-fixed, paraffin-embedded (FFPE) CMT tissue samples and compared ddPCR results with next-generation sequencing (NGS) data, achieving high concordance. Plasma and serum samples were also assessed for mutation concordance with tissue results. Our findings indicate a higher frequency of the PIK3CA (H1047R) mutations in benign and grade I malignant CMTs compared to more aggressive malignancies. The ddPCR assay demonstrated high sensitivity and specificity, with plasma testing showing 78.6% sensitivity and 87.5% specificity, and serum testing showing 66.7% sensitivity and 90.0% specificity. These results highlight the viability of liquid biopsy as a minimally invasive method for monitoring PIK3CA mutations in canine patients. The study suggests that liquid biopsy techniques hold significant promise for improving the early detection and monitoring of canine cancers, warranting further research to refine these methods and explore their applications in canine cancer diagnostics and treatment.

Abstract Background DNA methylation showed notable potential to act as a diagnostic marker in many cancers. Many studies proposed DNA methylation biomarker in OSCC detection, while most of these studies are limited to specific cohorts or geographical location. However, the generalizability of DNA methylation as a diagnostic marker in oral cancer across different geographical locations is yet to be investigated. Methods We used genome-wide methylation data from 384 oral cavity cancer and normal tissues from TCGA HNSCC and eastern India. The common differentially methylated CpGs in these two cohorts were used to develop an Elastic-net model that can be used for the diagnosis of OSCC. The model was validated using 812 HNSCC and normal samples from different anatomical sites of oral cavity from seven countries. Droplet Digital PCR of methyl-sensitive restriction enzyme digested DNA (ddMSRE) was used for quantification of methylation and validation of the model with 22 OSCC and 22 contralateral normal samples. Additionally, pyrosequencing was used to validate the model using 46 OSCC and 25 adjacent normal and 21 contralateral normal tissue samples. Results With ddMSRE, our model showed 91% sensitivity, 100% specificity, and 95% accuracy in classifying OSCC from the contralateral normal tissues. Validation of the model with pyrosequencing also showed 96% sensitivity, 91% specificity, and 93% accuracy for classifying the OSCC from contralateral normal samples, while in case of adjacent normal samples we found similar sensitivity but with 20% specificity, suggesting the presence of early disease methylation signature at the adjacent normal samples. Methylation array data of HNSCC and normal tissues from different geographical locations and different anatomical sites showed comparable sensitivity, specificity, and accuracy in detecting oral cavity cancer with across. Similar results were also observed for different stages of oral cavity cancer. Conclusions Our model identified crucial genomic regions affected by DNA methylation in OSCC and showed similar accuracy in detecting oral cancer across different geographical locations. The high specificity of this model in classifying contralateral normal samples from the oral cancer compared to the adjacent normal samples suggested applicability of the model in early detection.

Abstract Background Nipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N gene of NiV. Results Our RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads. Conclusions These findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices.

Ascochyta blight (AB) disease, caused by the fungal pathogen Ascochyta rabiei, is a major production constraint in many chickpea‐growing regions worldwide, causing substantial reductions in grain yield and seed quality. The management of AB is challenging due to limited genetic resistance and the evolving aggressiveness of A. rabiei. Currently, there is a heavy reliance on visual assessment by expert pathologists for the detection and quantification of disease severity, and limited ability to impartially quantify pathogen growth and inoculum potential in the field. In this study, we address these gaps by developing a single‐copy genetic marker for the sensitive detection and quantification of A. rabiei mycelium and conidiospores. Using a droplet digital PCR (ddPCR) assay, our method provides a sensitive (≤5 × 10−2 pg DNA, 1 gene copy) approach to assess A. rabiei biomass throughout its lifecycle on living and dead plant tissues. The method (i) has specificity to A. rabiei in diseased plant samples; (ii) discriminates among chickpea cultivars with varying AB resistance prior to the onset of visible symptoms; (iii) detects differences in primary A. rabiei conidiospore inoculum load from field‐grown chickpea stubble; and (iv) has potential application to disease management, breeding and epidemiology. [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]

Objective To establish a rapid detection method for invasive candidiasis based on droplet digital polymerase chain reaction (ddPCR). Methods We developed an assay system using a microtitre-based digital PCR platform and designed primer probes specific for four Candida species, namely Candida albicans, Candida smoothii, Candida near-smoothii, and Candida tropicalis. (1) The Limit of Blank (LOB) range and positive judgment value were determined by analyzing No Template Control (NTC) samples. (2) The Limit of Detection (LOD) range was determined by diluting positive samples with 10 replicate extractions at each concentration gradient. (3) The Linear Limit of Quantitation (LOQ) range was determined by repetitive testing of diluted samples. (4) The linear range limit was determined through gradient dilution of the positive samples. (5) The coefficient of variation (CV), calculated from the logarithmic values of the resultant concentrations, was assessed by extracting and testing positive samples in 12 repetitions at both high and low concentrations. (6) Method reliability was evaluated by calculating the CV from the logarithmic values of the resultant concentrations obtained from clinical samples with fungal culture results. Results The ddPCR assay detected Candida LOB at a range of 0 ~ 81 copies/mL, with a positive threshold set at 5 3 positive microdroplets. The LOD and LOQ were determined to be 3 x 10² copies/mL. The linear range for detecting different concentration gradients was found to be between 3 x 10² and 3 x 107 copies/mL, with high correlation coefficients observed for Candida albicans (R² = 0.999 5), Candida smoothii (R² = 0.998 9 ), Candida near-smoothii (R² = 0.999 4), and Candida tropicalis (R² = 0.999). Additionally, the coefficient of variation for the resultant concentration logarithmic values was less than 5%, meeting precision requirements. Furthermore, preliminary validation using clinical specimens demonstrated consistent results compared to clinical culture findings. Conclusion ddPCR exhibits rapidity, high sensitivity, good repeatability, and high specificity in detecting invasive candidiasis in critically ill patients. This study highlights the potential value of droplet digital PCR as a diagnostic tool for invasive candidiasis. [ABSTRACT FROM AUTHOR]

Objective: Cell‐free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR‐based approach allowing us to separately quantify absolute values of dd‐cfDNA and recipient‐derived cfDNA (rd‐cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. Methods: We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false‐negative and false‐positive rejections in six patients (five adults, one child). Results: There was a substantial discrepancy between relative (DF) and absolute cfDNA‐levels in several clinical situations. Rd‐ and dd‐cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. Conclusions: Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection. [ABSTRACT FROM AUTHOR]

Introduction: Donor leucocyte survival following red blood cell (RBC) transfusion, known as transfusion‐associated microchimerism (TAM), can occur in some patients. In Australia, despite the introduction of leucocyte filtration (leucodepletion) during RBC manufacture, TAM has been detected in adult trauma patients. However, the incidence of TAM in Australian pediatric patients has not been analyzed. Methods: Patients aged 0–16 years were recruited across two cohorts. Retrospective participants had RBC transfusion between January 1, 2002 and November 15, 2017 and prospective participants received RBC transfusion between December 1, 2016 and November 25, 2020. Twelve bi‐allelic insertion/deletion (InDel) polymorphisms were used to detect microchimerism amplification patterns using real‐time PCR (RT‐PCR) and droplet digital PCR (ddPCR). Results: Of the retrospective cohort (n = 40), six patients showed amplification of InDel sequences indicating potential microchimerism. For three patients, minor InDel sequences were detected using RT‐PCR only, two patients had minor InDel amplification using ddPCR only, and one patient had minor InDel amplification that was confirmed using both techniques. Amplification of minor sequences occurred in three patients who had received a bone marrow transplant in addition to RBC transfusion. In the prospective cohort (n = 25), no InDel amplification indicating potential microchimerism was detected using RT‐PCR. Discussion: Cell‐based therapies had been administered in three patients where microchimerism amplification patterns were detected. Three patients have microchimerism that may be attributed to RBC transfusion. In prospective patients, who received leucodepleted and gamma‐irradiated RBC units, no potential microchimerism amplification were detected. ddPCR may be a suitable technique for TAM analysis but requires further evaluation. [ABSTRACT FROM AUTHOR]

Pine wilt disease (PWD), which poses a significant risk to pine plantations across the globe, is caused by the pathogenic agent Bursaphelenchus xylophilus, also referred to as the pine wood nematode (PWN). A droplet digital PCR (ddPCR) assay was developed for the quick identification of the PWN in order to improve detection sensitivity. The research findings indicate that the ddPCR assay demonstrated significantly higher analysis sensitivity and detection sensitivity in comparison to traditional quantitative PCR (qPCR). However, it had a more limited dynamic range. High specificity was shown by both the ddPCR and qPCR techniques in the diagnosis of the PWN. Assessments of reproducibility revealed that ddPCR had lower coefficients of variation at every template concentration. Inhibition tests showed that ddPCR was less susceptible to inhibitors. There was a strong linear association between standard template measurements obtained using ddPCR and qPCR (Pearson correlation = 0.9317; p < 0.001). Likewise, there was strong agreement (Pearson correlation = 0.9348; p < 0.001) between ddPCR and qPCR measurements in the evaluation of pine wood samples. Additionally, wood samples from symptomatic (100% versus 86.67%) and asymptomatic (31.43% versus 2.9%) pine trees were diagnosed with greater detection rates using ddPCR. This study's conclusions highlight the advantages of the ddPCR assay over qPCR for the quantitative detection of the PWN. This method has a lot of potential for ecological research on PWD and use in quarantines. [ABSTRACT FROM AUTHOR]

Background: Nipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N gene of NiV. Results: Our RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads. Conclusions: These findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices. [ABSTRACT FROM AUTHOR]

Species of Saccharomyces genus have played an irreplaceable role in alcoholic beverage and baking industry for centuries. S. cerevisiae has also become an organism of choice for industrial production of alcohol and other valuable chemicals and a model organism shaping the rise of modern genetics and genomics in the past few decades. Today´s brewing industry faces challenges of decreasing consumption of traditional beer styles and increasing consumer demand for new styles, flavors and aromas. The number of currently used brewer's strains and their genetic diversity is yet limited and implementation of more genetic and phenotypic variation is seen as a solution to cope with the market challenges. This requires modification of current production strains or introduction of novel strains from other settings, e.g. industrial or wild habitats into the brewing industry. Due to legal regulation in many countries and negative customer perception of GMO organisms, the production of food and beverages requires non-GMO production organisms, whose development can be difficult and time-consuming. Here, we apply FIND-IT (Fast Identification of Nucleotide variants by DigITal PCR), an ultrafast genome-mining method, for isolation of novel yeast variants with varying flavor profiles. The FIND-IT method uses combination of random mutagenesis, droplet digital PCR with probes that target a specific desired mutation and a sub-isolation of the mutant clone. Such an approach allows the targeted identification and isolation of specific mutant strains with eliminated production of certain flavor and off-flavors and/or changes in the strain metabolism. We demonstrate that the technology is useful for the identification of loss-of function or gain of function mutations in unrelated industrial and wild strains differing in ploidy. Where no other phenotypic selection exists, this technology serves together with standard breeding techniques as a modern tool facilitating a modification of (brewer's) yeast strains leading to diversification of the product portfolio. • Mutagenesis and ddPCR allow for targeted mutant identification in brewer's yeast. • Modification of flavor profiles has been achieved in various industrial yeasts. • Heterothallic S. eubayanus strain facilitates development of novel lager hybrids. • Combination of the method with other non-GM techniques creates phenotypic diversity. • Method enables non-GMO engineering and improvement of brewing-related properties. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda (E. tarda) can infect humans and a variety of animals, including fish, amphibians, reptiles, birds, and mammals. However, a more highly sensitive, specific, and repeatable test for its detection is lacking. The objective of this study was to develop a highly sensitive, specific, and repeatable droplet digital polymerase chain reaction (ddPCR)-based method for the quantitative detection of E. tarda. The gyrB gene was selected as the target gene, and primers and probe were designed and synthesized. Using E. tarda genomic DNA as templates, the reaction method was optimized to establish a linear relationship with realtime PCR detection methods. The sensitivity, specificity, and repeatability of the method were analyzed, and clinical samples were tested. When the primer and probe concentrations were 900 and 300 nM, respectively, and the annealing temperature was 57°C, the efficiency of the ddPCR amplification reaction was highest and the boundary between positive and negative droplet distribution was clearest. The sensitivity was high, with detection limit being as low as 0.56 copies·µL-1; additionally, and a good linear relationship (R² = 0.9962) between ddPCR and real-time PCR detection, within the range of 1-25,000 copies·µL-1, was evident. The repeatability was good, with a detection coefficient of variation of 2.74%. There was no cross-reactivity with 15 other common pathogenic microorganisms in aquatic animals (Streptococcus agalactiae, Streptococcus iniae, Streptococcus suis type 2, Nocardia seriolae, Vibrio parahaemolyticus, Aeromonas sobria, red sea bream iridovirus, decapod iridescent virus 1, enterocytozoon hepatopenaei, carp edema virus, Koi herpesvirus, goldfish hematopoietic necrosis virus, tilapia lake virus, viral nervous necrosis virus, or grass carp reovirus) in positive samples. Among the 48 clinical samples, including Bahaba taipingensis and its live food fish, pond water samples, and routine monitoring samples (Koi), 21 were positive for E. tarda, consistent with the bacterial isolation and identification results. The E. tarda ddPCR detection method has high specificity, sensitivity, and repeatability, can more accurately quantify E. tarda, and provides a useful reference for research related to this bacterium. [ABSTRACT FROM AUTHOR]

Usher syndrome (USH) is an inherited disorder characterized by sensorineural hearing loss (SNHL), retinitis pigmentosa (RP)-related vision loss, and vestibular dysfunction. USH presents itself as three distinct clinical types, 1, 2, and 3, with no biomarker for early detection. This study aimed to explore whether microRNA (miRNA) expression in USH cell lines is dysregulated compared to the miRNA expression pattern in a cell line derived from a healthy human subject. Lymphocytes from USH patients and healthy individuals were isolated and transformed into stable cell lines using Epstein–Barr virus (EBV). DNA from these cell lines was sequenced using a targeted panel to identify gene variants associated with USH types 1, 2, and 3. Microarray analysis was performed on RNA from both USH and control cell lines using NanoString miRNA microarray technology. Dysregulated miRNAs identified by the microarray were validated using droplet digital PCR technology. DNA sequencing revealed that two USH patients had USH type 1 with gene variants in USH1B (MYO7A) and USH1D (CDH23), while the other two patients were classified as USH type 2 (USH2A) and USH type 3 (CLRN-1), respectively. The NanoString miRNA microarray detected 92 differentially expressed miRNAs in USH cell lines compared to controls. Significantly altered miRNAs exhibited at least a twofold increase or decrease with a p value below 0.05. Among these miRNAs, 20 were specific to USH1, 14 to USH2, and 5 to USH3. Three miRNAs that are known as miRNA-183 family which are crucial for inner ear and retina development, have been significantly downregulated as compared to control cells. Subsequently, droplet digital PCR assays confirmed the dysregulation of the 12 most prominent miRNAs in USH cell lines. This study identifies several miRNA signatures in USH cell lines which may have potential utility in Usher syndrome identification. [ABSTRACT FROM AUTHOR]

Background: DNA methylation showed notable potential to act as a diagnostic marker in many cancers. Many studies proposed DNA methylation biomarker in OSCC detection, while most of these studies are limited to specific cohorts or geographical location. However, the generalizability of DNA methylation as a diagnostic marker in oral cancer across different geographical locations is yet to be investigated. Methods: We used genome-wide methylation data from 384 oral cavity cancer and normal tissues from TCGA HNSCC and eastern India. The common differentially methylated CpGs in these two cohorts were used to develop an Elastic-net model that can be used for the diagnosis of OSCC. The model was validated using 812 HNSCC and normal samples from different anatomical sites of oral cavity from seven countries. Droplet Digital PCR of methyl-sensitive restriction enzyme digested DNA (ddMSRE) was used for quantification of methylation and validation of the model with 22 OSCC and 22 contralateral normal samples. Additionally, pyrosequencing was used to validate the model using 46 OSCC and 25 adjacent normal and 21 contralateral normal tissue samples. Results: With ddMSRE, our model showed 91% sensitivity, 100% specificity, and 95% accuracy in classifying OSCC from the contralateral normal tissues. Validation of the model with pyrosequencing also showed 96% sensitivity, 91% specificity, and 93% accuracy for classifying the OSCC from contralateral normal samples, while in case of adjacent normal samples we found similar sensitivity but with 20% specificity, suggesting the presence of early disease methylation signature at the adjacent normal samples. Methylation array data of HNSCC and normal tissues from different geographical locations and different anatomical sites showed comparable sensitivity, specificity, and accuracy in detecting oral cavity cancer with across. Similar results were also observed for different stages of oral cavity cancer. Conclusions: Our model identified crucial genomic regions affected by DNA methylation in OSCC and showed similar accuracy in detecting oral cancer across different geographical locations. The high specificity of this model in classifying contralateral normal samples from the oral cancer compared to the adjacent normal samples suggested applicability of the model in early detection. [ABSTRACT FROM AUTHOR]

Background: Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method – droplet digital PCR (ddPCR). Methods: Less than 5 mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR. Results: Thyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases. Conclusions: Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment. [ABSTRACT FROM AUTHOR]

Background: The CDKN2A gene is frequently affected by somatic copy number variations (SCNVs, including deletions and amplifications [SCNdel and SCNamp]) in the cancer genome. Using surgical gastric margin tissue samples (SMs) as the diploid reference in SCNV analysis via CDKN2A/P16-specific real-time PCR (P16-Light), we previously reported that the CDKN2A SCNdel was associated with a high risk of metastasis of gastric carcinoma (GC). However, the status of CDKN2A SCNVs in SMs and their clinical significance have not been reported. Methods: Peripheral white blood cell (WBC) and frozen GC and SM tissue samples were collected from patients (n = 80). Droplet digital PCR (ddPCR) was used to determine the copy number (CN) of the CDKN2A gene in tissue samples using paired WBCs as the diploid reference. Results: A novel P16-ddPCR system was initially established with a minimal proportion (or limit, 10%) of the detection of CDKN2A CN alterations. While CDKN2A SCNamp events were detected in both SMs and GCs, fewer CDKN2A SCNdel events were detected in SMs than in GCs (15.0% vs. 41.3%, P = 4.77E-04). Notably, significantly more SCNamp and fewer SCNdel of the CDKN2A gene were detected in SMs from GC patients without metastasis than in those from patients with lymph node metastasis by P16-ddPCR (P = 0.023). The status of CDKN2A SCNVs in SM samples was significantly associated with overall survival (P = 0.032). No cancer deaths were observed among the 11 patients with CDKN2A SCNamp. Conclusion: CDKN2A SCNVs in SMs identified by P16-ddPCR are prevalent and significantly associated with GC metastasis and overall survival. [ABSTRACT FROM AUTHOR]

Tuberculous pleurisy is one of the most common types of extra-pulmonary tuberculosis, but the sensitivity of conventional mycobacterial culture (Culture) or Xpert MTB/RIF assay (Xpert) is not satisfying. This multicentre cohort study evaluated the accuracy of a new cell-free DNA droplet digital PCR assay (cf-ddPCR) for diagnosing tuberculous pleurisy. Patients with suspected tuberculosis (≥5 years of age) with pleural effusion were consecutively recruited from nine research sites across six provinces in China between September 2020 to May 2022. Culture, Xpert, Xpert MTB/RIF Ultra assay (Ultra), real-time PCR, and cf-ddPCR were performed simultaneously for all specimens. A total of 321 participants were enrolled, and data from 281 (87.5%) participants were available, including 105 definite tuberculous pleurisy, 113 possible tuberculous pleurisy and 63 non-tuberculous pleurisy according to the composite reference standard. The sensitivity of cf-ddPCR was 90.5% (95/105, 95% CI, 82.8–95.1%) in the definite tuberculous pleurisy group, which was significantly higher than those of Culture (57.1%, 60/105, 95% CI, 47.1–66.6%, p < 0.001), Xpert (46.7%, 49/105, 95% CI, 37.0–56.6%, p < 0.001), Ultra (69.5%, 73/105, 95% CI, 59.7–77.9%, p < 0.001) and real-time PCR (75.2%, 79/105, 95% CI, 65.7–82.9%, p < 0.001). In possible tuberculous pleurisy, whose results of Culture and Xpert were both negative, the sensitivity of cf-ddPCR was 61.1% (69/113, 95% CI, 51.4–70.0%), which was still significantly higher than that of Ultra (27.4%, 31/113, 95% CI, 19.7–36.8%, p < 0.001) and real-time PCR (38.9%, 44/113, 95% CI, 30.0–48.6%, p < 0.001). The performance of cf-ddPCR is superior to Culture, Xpert, Ultra, and real-time PCR, indicating that improved diagnostic accuracy can be anticipated by incorporating this new assay. [ABSTRACT FROM AUTHOR]

Spelt products are popular with consumers achieving higher market prizes, making them susceptible to food adulteration with less valuable cereals. To facilitate product control, a recently developed duplex droplet digital PCR method enables the detection and quantification of common wheat (Triticum aestivum) contaminations in food products made from spelt (Triticum spelta). The duplex droplet digital PCR assay targets the γ-gliadin gene and the Q-locus of both subspecies. In this study, the method was validated in an interlaboratory ring trial involving 11 participating laboratories. Test materials containing defined proportions of spelt and common wheat were prepared and tested. The ring trial procedure included DNA extraction of the test samples and determination of subspecies proportions using droplet digital PCR. Results from the ring trial confirmed the method's capability for specific detection and quantification of common wheat in spelt, with acceptable relative measurement uncertainties, and without requiring reference material for calibration. To our knowledge, this is the first interlaboratory validation of a digital PCR method for species differentiation in food. [ABSTRACT FROM AUTHOR]

Advanced diagnostic technologies have made accurate and precise diagnosis of pathogens easy. Herein, we present a new diagnostic method, droplet digital PCR (ddPCR), to detect and quantify Acinetobacter baumannii in mini bronchoalveolar lavage (mini-BAL) samples. A. baumannii causes ventilator-associated pneumonia (VAP), a severe healthcare infection affecting patients' lungs. VAP carries a high risk of morbidity and mortality, making its timely diagnosis crucial for prompt and effective management. Methodology. The assay performance was evaluated by comparing colonization data, quantitative culture results, and different generations of PCR (traditional PCR and Real-Time PCR—qPCR Taqman® and SYBR® Green). The ddPCR and qPCR Taqman® prove to be more sensitive than other molecular techniques. Reasonable analytical specificity was obtained with ddPCR, qPCR TaqMan®, and conventional PCR. However, qPCR SYBR® Green technology presented a low specificity, making the results questionable in clinical samples. DdPCR detected/quantified A. baumanni in more clinical samples than other methods (38.64% of the total samples). This emerging ddPCR technology offers promising advantages such as detection by more patients and direct quantification of pathogens without calibration curves. [ABSTRACT FROM AUTHOR]

Pathogenic viruses in environmental water are usually present in levels too low for direct detection and thus, a concentration step is often required to increase the analytical sensitivity. The objective of this study was to evaluate an automated filtration device, the Innovaprep Concentrating Pipette Select (CP Select) for the rapid concentration of viruses in saline water samples, while considering duration of process and ease of use. Four bacteriophages (MS2, P22, Phi6, and PhiX174) and three animal viruses (adenovirus, coronavirus OC43, and canine distemper virus) were seeded in artificial seawater, aquarium water, and bay water samples, and processed using the CP Select. The recovery efficiencies of viruses were determined either using a plaque assay or droplet digital PCR (ddPCR). Using plaque assays, the average recovery efficiencies for bacteriophages ranged from 4.84 ± 3.8% to 82.73 ± 27.3%, with highest recovery for P22 phage. The average recovery efficiencies for the CP Select were 39.31 ± 26.6% for adenovirus, 19.04 ± 11.6% for coronavirus OC43, and 19.84 ± 13.6% for canine distemper virus, as determined by ddPCR. Overall, viral genome composition, not the size of the virus, affected the recovery efficiencies for the CP Select. The small sample volume size used for the ultrafilter pipette of the system hinders the use of this method as a primary concentration step for viruses in marine waters. However, the ease of use and rapid processing time of the CP Select are especially beneficial when rapid detection of viruses in highly contaminated water, such as wastewater or sewage-polluted surface water, is needed. [ABSTRACT FROM AUTHOR]

During the last few years, cell-free DNA (cfDNA) has emerged as a possible noninvasive biomarker for prediction of complications after lung transplantation. We previously published a proof-of-concept study using a digital droplet polymerase chain reaction (ddPCR)-based method for detection of cfDNA. In the current study, we aimed to further evaluate the potential clinical usefulness of detecting chronic lung allograft dysfunction (CLAD) using three different ddPCR applications measuring and calculating the donor fraction (DF) of cfDNA as well as one method using the absolute amount of donor-derived cfDNA. We analyzed 246 serum samples collected from 26 lung transplant recipients. Nine of the patients had ongoing CLAD at some point during follow-up. All four methods showed statistically significant elevation of the measured variable in the CLAD samples compared to the non-CLAD samples. The results support the use of ddPCR-detected cfDNA as a potential biomarker for prediction of CLAD. These findings need to be validated in a subsequent prospective study. [ABSTRACT FROM AUTHOR]

Accurate and early diagnosis of monkeypox virus (MPXV) is crucial for controlling epidemics and treating affected individuals promptly. This study aimed to assess the analytical and clinical performance of the MolecisionTM Monkeypox Virus qPCR Assay, Biorain Monkeypox Virus ddPCR Assay, and MAGLUMI® Monkeypox Virus Ag (chemiluminescence immunoassay, CLIA) Assay. Additionally, it aimed to compare the clinical application of antigen and nucleic acid assays to offer insights into using commercial monkeypox assay kits. Specimens from 117 clinical patients, serial diluted virus cell culture supernatant, and artificially created positive samples were tested to evaluate the performance of these assay kits for MPXV diagnostics. The Biorain Monkeypox Virus ddPCR Assay had a limit of detection (LoD) of 3.89 CCID50/mL, while the MolecisionTM Monkeypox Virus qPCR Assay had an LoD of 15.55 CCID50/mL. The MAGLUMI® Monkeypox Virus Ag (CLIA) Assay had an LoD of 0.500 pg/mL. The accuracy of the MolecisionTM Monkeypox Virus qPCR Assay was comparable to the Biorain Monkeypox Virus ddPCR Assay, and the MAGLUMI® Monkeypox Virus Ag (CLIA) Assay demonstrated high sensitivity. The specificity of all three MPXV diagnostic assays for clinical specimens with potential cross-reacting substances was 100%. In conclusion, this study provides valuable insights into the clinical application of monkeypox assays, supporting efforts to mitigate and control the spread of monkeypox. [ABSTRACT FROM AUTHOR]