Donor Fractions of Cell-Free DNA Are Elevated During CLAD But Not During Infectious Complications After Lung Transplantation.

During the last few years, cell-free DNA (cfDNA) has emerged as a possible non-invasive 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.

Detection of residual pluripotent stem cells in cell therapy products utilizing droplet digital PCR: an international multisite evaluation study.

The presence of residual undifferentiated pluripotent stem cells (PSCs) in PSC-derived cell therapy products (CTPs) is a major safety issue for their clinical application, due to the potential risk of PSC-derived tumor formation. An international multidisciplinary multisite study to evaluate a droplet digital PCR (ddPCR) approach to detect residual undifferentiated PSCs in PSC-derived CTPs was conducted as part of the Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee. To evaluate the use of ddPCR in quantifying residual iPSCs in a cell sample, different quantities of induced pluripotent stem cells (iPSCs) were spiked into a background of iPSC-derived cardiomyocytes (CMs) to mimic different concentrations of residual iPSCs. A one step reverse transcription ddPCR (RT-ddPCR) was performed to measure mRNA levels of several iPSC-specific markers and to evaluate the assay performance (precision, sensitivity, and specificity) between and within laboratories. The RT-ddPCR assay variability was initially assessed by measuring the same RNA samples across all participating facilities. Subsequently, each facility independently conducted the entire process, incorporating the spiking step, to discern the parameters influencing potential variability. Our results show that a RT-ddPCR assay targeting ESRG, LINC00678, and LIN28A genes offers a highly sensitive and robust detection of impurities of iPSC-derived CMs and that the main contribution to variability between laboratories is the iPSC-spiking procedure, and not the RT-ddPCR. The RT-ddPCR assay would be generally applicable for tumorigenicity evaluation of PSC-derived CTPs with appropriate marker genes suitable for each CTP.

Multiplex droplet digital PCR for 22q11.2 microdeletions screening and DiGeorge syndrome diagnostics.

BACKGROUND AND AIMS: DiGeorge syndrome (DGS) is a genetic disorder manifesting in polymorphic symptoms related to developmental abnormalities of various organs including thymus. DGS is caused by microdeletions in the 22q11.2 region between several low copy repeats (LCR) occurring in approximately 1 in 4000 live births. Diagnosis of DGS relies on phenotypic examination, qPCR, ultrasound, FISH, MLPA and NGS which can be relatively inaccurate, time-consuming, and costly. MATERIALS AND METHODS: A novel multiplex droplet digital PCR (ddPCR) assay was designed, optimized and validated for detection and mapping 22q11.2 microdeletions by simultaneous amplification of three targets - TUPLE1, ZNF74, D22S936 - within the deletion areas and one reference target - RPP30 - as an internal control. RESULTS: The assay reliable identified microdeletions when the template concentration was >32 copies per reaction and successfully detected LCR22A-B, LCR22A-C, LCR22A-D, and LCR22B-C deletions in clinical samples from 153 patients with signs of immunodeficiency. In patients with the microdeletions, flow cytometry detected a significant increase in B-cell and natural killer cell counts and percentages, while T-cell percentages and T-cell receptor excision circle (TREC) numbers decreased. CONCLUSION: The designed ddPCR assay is suitable for diagnosing DGS using whole blood and blood spots.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Quadruplex Droplet Digital PCR Assay for Screening and Quantification of SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by the rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since early 2020, has highlighted the need for sensitive and reliable diagnostic methods. Droplet digital PCR (ddPCR) has demonstrated superior performance over the gold-standard reverse transcription PCR (RT-PCR) in detecting SARS-CoV-2. In this study, we explored the development of a multiplex ddPCR assay that enables sensitive quantification of SARS-CoV-2, which could be utilized for antiviral screening and the monitoring of COVID-19 patients. We designed a quadruplex ddPCR assay targeting four SARS-CoV-2 genes and evaluated its performance in terms of specificity, sensitivity, linearity, reproducibility, and precision using a two-color ddPCR detection system. The results showed that the quadruplex assay had comparable limits of detection and accuracy to the simplex ddPCR assays. Importantly, the quadruplex assay demonstrated significantly improved performance for samples with low viral loads and ambiguous results compared to the standard qRT-PCR approach. The developed multiplex ddPCR represents a valuable alternative and complementary tool for the diagnosis of SARS-CoV-2 and potentially other pathogens in various application scenarios beyond the current COVID-19 pandemic. The improved sensitivity and reliability of this assay could contribute to more effective disease monitoring and antiviral screening during the ongoing public health crisis.

Discovery unbalanced DNA mixtures and evaluation mixing ratio via a droplet digital PCR method.

BACKGROUND: Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator's conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures. METHODS: The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios. RESULTS: The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately. CONCLUSIONS: A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

A Comparative Evaluation of Three Diagnostic Assays for the Detection of Human Monkeypox.

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.

A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications.

In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30, and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies.

Evaluation of an Automated Ultrafiltration System for Concentrating a Range of Viruses from Saline Waters.

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.

To evaluate the performance of simultaneous amplification and testing assay for group B Streptococcus detection: comparison with real-time PCR and ddPCR assays.

BACKGROUND: To evaluate the performance of simultaneous amplification and testing (SAT) assay for the detection of group B Streptococcus (GBS) in maternal vaginal and perianal swabs compared with real-time polymerase chain reaction (RT-PCR). METHODS: We obtained vaginal and perianal swabs from 1474 pregnant women at the Obstetrics and Gynecology Hospital of Fudan University (Shanghai, China) between April 2023 and June 2023. Vaginal and perianal swabs were collected at 35-37 weeks of gestation. Swabs were tested for GBS simultaneously by using the SAT assay and RT-PCR, and a comparative analysis (kappa coefficient) was performed. Furthermore, we conducted additional droplet digital PCR (ddPCR) tests to confirm the results when there were controversial results between SAT and RT-PCR. In addition, we compared the limit of detection, technical specificity, repeatability and reproducibility of SAT-GBS with those of routine RT-PCR assays. RESULTS: In our study, the detection rate of clinical GBS according to the SAT assay was 11.5% (169/1471). The SAT assay showed a sensitivity of 91.8%, a specificity of 99.9%, a diagnostic accuracy of 98.9%, a positive predictive value (PPV) of 99.4% and a negative predictive value (NPV) of 98.8%. The kappa value between RT-PCR and SAT was 0.917. CONCLUSIONS: This SAT assay for the detection of group B Streptococcus is not only easy to perform but can also detect GBS sensitively and specifically and may be used in the regular molecular diagnosis of GBS infection among pregnancies.

Evaluating storage conditions and enhancement strategies on viral biomarker recovery for WBE applications.

Wastewater-based epidemiology (WBE) is a valuable disease surveillance tool. However, little is known on how factors such as transportation, storage, and wastewater characteristics influence the accuracy of the quantification methods. Hence, this study investigated the impact of storage temperatures and physicochemical characteristics of wastewater on SARS-CoV-2 and influenza A stability using droplet digital PCR. Additionally, strategies to enhance viral recovery were explored. Municipal influent wastewater stored between ±25 and -80 °C was assessed for a period of 84 days to determine viral degradation. Degradation up to 94.1% of influenza A and SARS-CoV-2 was observed in all samples with the highest at ±25 °C. Viral degradation was correlated to the changes in wastewater physicochemical characteristics. The low degradation observed of SARS-CoV-2 in the spiked pellets were indicative of viral adhesion to wastewater solids, which correlated with changes in pH. Ultrasonication frequencies ranging from 4 to 16 kHz, increased SARS-CoV-2 concentrations in the supernatant between 3.30 and 35.65%, indicating viral RNA attachment to wastewater solids. These results highlight the importance of additional pretreatment methods for maximizing RNA recovery from wastewater samples. Based on these findings, it was deduced that wastewater preservation studies are essential, and pretreatment should be included in the WBE methodology.

Quantitative detection and survival analysis of VBNC Salmonella Typhimurium in flour using droplet digital PCR and DNA-intercalating dyes.

The difficulty in detecting viable but non-culturable (VBNC) Salmonella by culture-dependent methods poses a risk to food safety. In our study, we applied a viability test to Salmonella following a lethal treatment and to flour samples inoculated with Salmonella to evaluate the effectiveness of viability polymerase chain reaction (PCR). Our findings revealed that the combination of both ddPCR and qPCR with those DNA-intercalating dyes could quantify viable cells at low concentrations when the plate counting method failed to detect them post-inactivation. Prolonged UV exposure did not induce cell membrane disruption, as confirmed with PMA-ddPCR, with insignificant differences in gene copies. However, samples exposed to DyeTox13 and DyeTox13 + EMA showed lower gene copy numbers, implying that enzymatic activity was decreased by UV exposure duration. In addition, temperature-dependent survival in flour revealed uniform decay rates and D values (time required for a 1 log reduction) of DNA in untreated samples across various temperatures. By contrast, different decay rates were observed with DNA-intercalating dyes (DyeTox13 and DyeTox13 + EMA), showing faster metabolic activity loss at higher temperatures in flour. The decay rates and D values, determined through plate counting and those DNA-intercalating dyes, indicated the potential presence of VBNC Salmonella. A strong correlation between DyeTox13 dyes and the plate counting method suggested DyeTox13 as a rapid alternative for detecting Salmonella in flour. The ddPCR with DNA-intercalating dyes could effectively evaluate Salmonella viability, facilitating more precise monitoring of VBNC in food. IMPORTANCE: Salmonella, a major foodborne pathogen, poses significant risks, particularly to vulnerable groups like infants, older people, and the immunocompromised. Accurate detection is vital for public health and food safety, given its potential to cause severe and life-threatening symptoms. Our study demonstrated digital polymerase chain reaction (ddPCR) with DNA-intercalating dyes for identifying the different physiological statuses of Salmonella. Also, the application of ddPCR with DNA-intercalating dyes offers quantification of viable cells post-disinfection as an alternative method in food. Utilizing ddPCR and DNA-intercalating dyes, we enhanced the detection of VBNC Salmonella, a form often undetectable by conventional methods. This innovative approach could significantly improve the precision and efficiency of detection for viable Salmonella. By providing deeper insights into its transmission potential, our method is a critical tool in preventing outbreaks and ensuring the safety of food products. This research contributes substantially to global efforts in controlling foodborne illnesses and safeguarding public health.

Development of a multiplex PCR assay and quantification of microbial markers by ddPCR for identification of saliva and vaginal fluid.

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10-9 ng/µl) and S. salivarius (2.5 ×10-6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10-4 ng/µl) and 0.48 copies/µl (2.5 ×10-7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.

Exosomal mRNA in plasma serves as a predictive marker for microvascular invasion in hepatocellular carcinoma.

BACKGROUND AND AIM: There is a pressing need for non-invasive preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC). This study investigates the potential of exosome-derived mRNA in plasma as a biomarker for diagnosing MVI. METHODS: Patients with suspected HCC undergoing hepatectomy were prospectively recruited for preoperative peripheral blood collection. Exosomal RNA profiling was conducted using RNA sequencing in the discovery cohort, followed by differential expression analysis to identify candidate targets. We employed multiplexed droplet digital PCR technology to efficiently validate them in a larger sample size cohort. RESULTS: A total of 131 HCC patients were ultimately enrolled, with 37 in the discovery cohort and 94 in the validation cohort. In the validation cohort, the expression levels of RSAD2, PRPSAP1, and HOXA2 were slightly elevated while CHMP4A showed a slight decrease in patients with MVI compared with those without MVI. These trends were consistent with the findings in the discovery cohort, although they did not reach statistical significance (P > 0.05). Notably, the expression level of exosomal PRPSAP1 in plasma was significantly higher in patients with more than 5 MVI than in those without MVI (0.147 vs 0.070, P = 0.035). CONCLUSION: This study unveils the potential of exosome-derived PRPSAP1 in plasma as a promising indicator for predicting MVI status preoperatively.

Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG.

Introduction: Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge. Methods: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively. Results: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively. Discussion: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

Detection of Foodborne RNA Viruses by Reverse Transcriptase Droplet Digital PCR.

Foodborne viruses remain the largest cause of human gastroenteritis and one of the largest contributors to foodborne illnesses worldwide. Currently, quantitative reverse transcription PCR (qRT-PCR) or real-time qPCR are the detection methods commonly used for quantification of foodborne viruses, but those methods have several disadvantages, such as relying on standard curves for quantification and the background noise from a bulk reaction. ddPCR uses an oil-water emulsion to form multiple droplets that partition small amounts of viral genetic material (DNA or RNA) into each of the droplets. These droplets then undergo amplification cycles and are analyzed using Poisson distributions. This allows for absolute quantification without the need for a standard curve, which makes ddPCR a precise tool in surveillance of foodborne viruses. Herein, we describe the process of detecting foodborne viruses using RNA isolated from various matrices. Up to 96 samples including the positive and negative controls can be analyzed on a single plate by ddPCR.

Droplet digital PCR analysis of CDH13 methylation status in Slovak women with invasive ductal breast cancer.

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR- versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

Pilot Study by Liquid Biopsy in Gastrointestinal Stromal Tumors: Analysis of PDGFRA D842V Mutation and Hypermethylation of SEPT9 Presence by Digital Droplet PCR.

Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In this pilot study, we advocate for droplet digital PCR (ddPCR) to diagnose GIST in tissue samples and explore its potential for early diagnosis via liquid biopsy, focusing on the PDGFRA D842V mutation and SEPT9 hypermethylated gene. We utilized ddPCR to analyze the predominant PDGFRA mutation (D842V) in surgical tissue samples from 15 GIST patients, correlating with pathologists' diagnoses. We expanded our analysis to plasma samples to compare DNA alterations between tumor tissue and plasma, also investigating SEPT9 gene hypermethylation. We successfully detected the PDGFRA D842V mutation in GIST tissues by ddPCR. Despite various protocols to enhance mutation detection in early-stage disease, it remained challenging, likely due to the low concentration of DNA in plasma samples. Additionally, the results of Area Under the Curve (AUC) for the hypermethylated SEPT9 gene, analyzing concentration, ratio, and abundance were 0.74 (95% Confidence Interval (CI): 0.52 to 0.97), 0.77 (95% CI: 0.56 to 0.98), and 0.79 (95% CI: 0.59 to 0.99), respectively. As a rare disease, the early detection of GIST through such biomarkers is particularly crucial, offering significant potential to improve patient outcomes.

Clinical evaluation of droplet digital PCR in the early identification of suspected sepsis patients in the emergency department: a prospective observational study.

Background: Rapid and accurate diagnosis of the causative agents is essential for clinical management of bloodstream infections (BSIs) that might induce sepsis/septic shock. A considerable number of suspected sepsis patients initially enter the health-care system through an emergency department (ED), hence it is vital to establish an early strategy to recognize sepsis and initiate prompt care in ED. This study aimed to evaluate the diagnostic performance and clinical value of droplet digital PCR (ddPCR) assay in suspected sepsis patients in the ED. Methods: This was a prospective single-centered observational study including patients admitted to the ED from 25 October 2022 to 3 June 2023 with suspected BSIs screened by Modified Shapiro Score (MSS) score. The comparison between ddPCR and blood culture (BC) was performed to evaluate the diagnostic performance of ddPCR for BSIs. Meanwhile, correlative analysis between ddPCR and the inflammatory and prognostic-related biomarkers were conducted to explore the relevance. Further, the health economic evaluation of the ddPCR was analyzed. Results: 258 samples from 228 patients, with BC and ddPCR performed simultaneously, were included in this study. We found that ddPCR results were positive in 48.13% (103 of 214) of episodes, with identification of 132 pathogens. In contrast, BC only detected 18 positives, 88.89% of which were identified by ddPCR. When considering culture-proven BSIs, ddPCR shows an overall sensitivity of 88.89% and specificity of 55.61%, the optimal diagnostic power for quantifying BSI through ddPCR is achieved with a copy cutoff of 155.5. We further found that ddPCR exhibited a high accuracy especially in liver abscess patients. Among all the identified virus by ddPCR, EBV has a substantially higher positive rate with a link to immunosuppression. Moreover, the copies of pathogens in ddPCR were positively correlated with various markers of inflammation, coagulation, immunity as well as prognosis. With high sensitivity and specificity, ddPCR facilitates precision antimicrobial stewardship and reduces health care costs. Conclusions: The multiplexed ddPCR delivers precise and quantitative load data on the causal pathogen, offers the ability to monitor the patient's condition and may serve as early warning of sepsis in time-urgent clinical situations as ED. Importance: Early detection and effective administration of antibiotics are essential to improve clinical outcomes for those with life-threatening infection in the emergency department. ddPCR, an emerging tool for rapid and sensitive pathogen identification used as a precise bedside test, has developed to address the current challenges of BSI diagnosis and precise treatment. It characterizes sensitivity, specificity, reproducibility, and absolute quantifications without a standard curve. ddPCR can detect causative pathogens and related resistance genes in patients with suspected BSIs within a span of three hours. In addition, it can identify polymicrobial BSIs and dynamically monitor changes in pathogenic microorganisms in the blood and can be used to evaluate antibiotic efficacy and survival prognosis. Moreover, the copies of pathogens in ddPCR were positively correlated with various markers of inflammation, coagulation, immunity. With high sensitivity and specificity, ddPCR facilitates precision antimicrobial stewardship and reduces health care costs.