Plasma DNA Research Articles

Behavioral variant frontotemporal dementia (bvFTD) and primary psychiatric disorders (PPD) have symptomatic overlap that leads to diagnostic challenges. Cell-free DNA (cfDNA) tests have revolutionized prenatal non-invasive testing and cancer diagnostics. This study investigated the diagnostic potential of brain-derived cfDNA in plasma to differentiate sporadic bvFTD from PPD subjects. Targeted bisulfite sequencing was conducted to quantify glial and neuronal cfDNA levels in plasma samples from 179 bvFTD and 102 PPD subjects of the multi-center DIPPA-FTD study. No significant differences were observed in the absolute levels of glial or neuronal cfDNA between the groups. However, the neuronal-to-glial cfDNA ratio (NGR) was significantly higher in PPD cases (p = 0.0002), suggesting a relative increase in neuronal cfDNA in PPD compared to bvFTD. Diagnostic performance analysis revealed that neuronal cfDNA and NGR achieved an area under the curve (AUC) of 0.74, with a sensitivity of 90% but a specificity of 44% in distinguishing bvFTD from PPD. While increased serum levels of neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) have been shown to effectively differentiate bvFTD from PPD, the addition of brain-derived cfDNA did not provide any incremental diagnostic benefit over these established biomarkers.

Invasive fungal disease (IFD) is a major cause of morbidity and mortality in the immunocompromised population. Early diagnosis is challenging due to the low sensitivity and non-specificity of non-invasive fungal biomarkers, the need for invasive specimen collection, and the limitations of culture and histopathology. Detection of circulating fungal cell-free DNA (cfDNA) in plasma and serum by polymerase chain reaction (PCR) represents a novel testing modality for rapid and accurate diagnosis of IFD. In this review, we summarize the performance characteristics of fungal cfDNA PCR for the diagnosis of invasive aspergillosis, mucormycosis, and Pneumocystis pneumonia. We discuss a testing algorithm that incorporates fungal cfDNA and the added diagnostic value of invasive specimen collection when non-invasive mold cfDNA PCR is performed first. Lastly, we discuss the role of diagnostic stewardship in fungal cfDNA PCR testing.

Abstract Background and Aims Cytomegalovirus (CMV) infection is the primary infectious complication in kidney transplantation (KT), and it contributes to morbidity and mortality rates. CMV-seropositive solid organ transplant recipients have a relatively lower risk of CMV infection when compared to CMV-seronegative recipients who receive allograft from CMV-seropositive donors. Nonetheless, some patients remain at risk. Our aim was to assess the association between the anti-CMV IgG titer and the risk of post-transplant CMV infection among CMV-seropositive KT recipients. Method We conducted a retrospective single-center study on adult CMV-seropositive KT recipients between January 2018 and June 2023. Clinical characteristics, risk factors, and outcomes were extracted from patient medical records. Patients who received anti-thymocyte globulin as induction therapy were given valganciclovir prophylaxis for 6 months post-transplant. The cumulative incidence of CMV infection was estimated using the Kaplan-Meier method (defined as detection of CMV DNA in plasma regardless of symptoms). We analyzed CMV-IgG as a predictor of CMV infection using Cox proportional hazards model (SPSS). The study was approved by the Institutional Review Board. Results The medical records of a total of 263 KT patients were retrieved, and a total of 27 were CMV-seronegative. A total of 236 CMV-seropositive KT recipients were included, being 66.1% male. Their mean ± SD age was 52.5 ± 11.9 years. Among these, 94% received deceased-donor allograft, of which 8.1% were after circulatory death donors (DCD), and 50.7% were expanded criteria donors. The median [IQR] mismatch was 4 [3.0, 5.0]. Regarding induction therapy, 62.7% of KT recipients received anti-thymocyte globulin. During a mean follow-up of 19.9 months, the cumulative incidence of CMV infection was 61.6% (Fig. 1). Lower titers of pretransplant CMV-IgG were associated with CMV infection (P = 0.031). Conclusion We observed that a lower pretransplant anti-CMV IgG titer is associated with an increased risk of post-transplant CMV infection among CMV-seropositive KT recipients. The universally available test for anti-CMV IgG titer could potentially stratify individuals at risk and target them to receive a more specific preventive strategy.

Abstract Background and Aims Plasma cell-free DNA (cfDNA) is a circulating extracellular DNA fragments and originates from inflammation and tissue damage. High levels of cfDNA have been reported in many clinical conditions, including CKD, hemodialysis and peritoneal dialysis (PD). In a recent work, we demonstrated that PD-related peritonitis induces cfDNA production. Unfortunately, we are unable to directly address the mechanism involved in cfDNA origin. The first aim of this study was confirmed the variation in plasma cfDNA after an episode of peritonitis in PD patients. The second purpose of this study was to elucidate the putative causative mechanism involved in cfDNA formation during peritonitis. In particular, we hypothesized that cfDNA production could be associated with apoptotic event and we investigated which pathway is involved in this process. Method We enrolled 54 PD patients and we divided them into 3 different groups: 25 PD patients without any history of peritonitis, 21 PD patients whose last episode of peritonitis was more than 3 months prior to enrollment, and 8 patients who had an episode of peritonitis within the 3 months prior to enrollment. CfDNA was extracted from plasma and was quantified by Real time PCR. Subsequent, analysis of apoptosis was qualitatively performed by DNA Ladder kit and quantitatively measured by ELISA tests for Caspase-3 (effector caspase), Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway). Results Quantitative analysis of cfDNA showed significantly higher levels in patients who had an episode of peritonitis compared with PD patients without any history of peritonitis. This data confirmed our previously results. Firstly, qualitative analysis of cfDNA showed higher DNA ladder formations, suggesting presence of apoptotic events (Fig. 1). The increase of apoptotic events was confirmed by Caspase-3 activation (P < 0.01) (Fig. 2). In particular, the levels of Caspase-3 was significantly higher in patients with acute peritonitis within the last 3 months. Notably, a significant correlation was observed between cfDNA and Caspase-3 levels (Spearman’s rank correlation coefficient = 0.511, P < 0.01) (Fig. 3). On the contrary, we observed lower levels of cfDNA and Caspase-3 in patients with a longer peritonitis-free period. In addition, we observed a strong significant correlation between Caspase-3 and Caspase-9 levels (Spearman’s rank correlation coefficient = 0.66, P < 0.01) and a significant correlation between Caspase-3 and Caspase-8 (Spearman’s rank correlation coefficient = 0.43, P = 0.037). Conclusion Based on our results, we confirmed that cfDNA is increased in the plasma of PD patients with recent peritonitis and we elucidated that cell apoptosis is one of the potential sources of cfDNA. Furthermore, we noticed dual apoptotic pathway activation: the intrinsic and extrinsic pathways converged on Caspase-3 increasing apoptotic events and production of cfDNA in PD patients. In particular, cfDNA, apototic events and dual pathway activation were enhanced in PD patients with acute peritonitis within the last 3 months.

Effects of seminal plasma cholesterol and androgen levels on breeding rooster sperm motility in vitro.

This research explored the effects of different concentrations of p-coumaric acid (PCA) on the quality of frozen-thawed boar semen. Boar sperm samples were pre-treated with different concentrations of PCA (0, 30, 60, 90, 120 μg/mL) prior to the freezing process. Subsequently, multiple parameters were analyzed post-freeze-thawing, including sperm morphological and kinetic characteristics, acrosome and membrane integrity, mitochondrial function, DNA integrity, antioxidant enzyme activities, the expression levels of the BCL-2, BAX, and Caspase-3 proteins, the in vitro fertilization rate of porcine oocytes, and the embryo cleavage rate. The findings indicated that, compared with the control group, the addition of 90 μg/mL PCA led to significant improvements in several key aspects. Sperm motility, average path velocity, straight-line velocity, curvilinear velocity, and beat cross frequency were all notably enhanced. Moreover, parameters related to sperm quality, such as acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity, also showed significant increases (all p < 0.05). In terms of antioxidant capacity, the 90 μg/mL PCA treatment significantly elevated the total antioxidant capacity, as well as the activities of superoxide dismutase, glutathione peroxidase, and catalase. Simultaneously, it caused a significant reduction in the contents of malondialdehyde and hydrogen peroxide (p < 0.05). Regarding protein expression, the addition of 90 μg/mL PCA significantly upregulated the expression level of the BCL-2 protein, while downregulating the relative expression levels of BAX and Caspase-3 (p < 0.05). Additionally, this concentration of PCA significantly improved the in vitro fertilization rate of porcine oocytes and the embryo cleavage rate (p < 0.05). In conclusion, incorporating PCA into the semen extender can potentially be advantageous for the cryopreservation of boar sperm, with 90 μg/mL being the optimal concentration.

Taxanes are life-prolonging treatments for patients with advanced prostate cancer. However, treatment resistance and lethal disease invariably develop. We here used liquid biopsies to identify and characterize resistance to cabazitaxel. We analysed serial plasma from metastatic castration-resistant prostate cancer patients treated with cabazitaxel in a prospective biomarker study (NCT03381326, N=97). Circulating tumor DNA (ctDNA) was studied using a bespoke, targeted genomic test (PCF_SELECT). Clinical and molecular variables were evaluated for associations with overall survival (OS) and radiographic progression free survival (rPFS). Patients categorized by median ctDNA fraction had progressively worse survival for ctDNA-negative versus low versus high (median OS: 26.8, 12.4, 8.2 months; median rPFS: 8.0, 5.3, 3.1 months). A ctDNA fraction increase at cycle 3 compared to patients who remained ctDNA negative associated with shorter OS (median 7.5 versus 29.9 months, HR, 4.60 [95% CI, 2.06-10.28], p<0.0001) and rPFS (median 2.6 versus 8.2 months, HR 3.73 [95% CI, 1.75-7.93], p<0.0001). Plasma DNA collected at progression on an androgen receptor pathway inhibitor was enriched for alterations in the androgen receptor (AR) gene whilst, after a taxane (docetaxel pre-cabazitaxel or post-cabazitaxel), there was enrichment of copy number gains of genes sited on chromosome 3 (ATR, PIK3CB, MLH1,FANCD2) or involved in cell cycle regulation, including mutually exclusive alterations in CCND1 and CDKN1B. Sequential liquid biopsy identifies ctDNA features associated with treatment benefit in mCRPC patients treated with cabazitaxel. There was overlap of gene alterations selected for at progression on docetaxel or cabazitaxel, in part explaining cross-resistance.

BackgroundBisulfite conversion is considered the gold standard for DNA methylation analysis, but it damages DNA and performs sub-optimally with clinical samples (e.g., formalin-fixed paraffin-embedded and circulating free plasma DNA (cfDNA)). Here we describe a comprehensive comparison of bisulfite and enzymatic methylation sequencing, using commercially available assays in clinically relevant patient samples and cell lines. We also report the first clinical enzymatic whole genome methylation sequencing (WGMS) in a cohort of patients with chronic lymphocytic leukemia (CLL). We report data from a multi-arm experiment comprising controlled reference material and clinically relevant samples to assess technical differences between enzymatic and chemical methylation conversion technologies.ResultsEnzymatic methylation sequencing was highly concordant to bisulfite data but outperformed bisulfite conversion in key sequencing metrics; the enzymatic method demonstrated significantly higher estimated counts of unique reads, reduced DNA fragmentation, and higher library yields than bisulfite conversion. Enzymatic conversion produced inferior methylation array data. Although bisulfite and enzymatic methods were highly concordant, the increased quality of multiple sequencing metrics seen in the enzymatic method enabled the development of robust clinical sample pipelines including targeted sequencing in cfDNA.ConclusionsUsing the enzymatic methylation sequencing methods described, we report a putative link of interleukin (IL)-15 methylation changes to acalabrutinib treatment response in a CLL clinical trial cohort (ACE-CL-001 trial, NCT02029443).Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-025-01959-0.

ABSTRACT Background Endocarditis is an important manifestation of extra-intestinal Whipple’s disease. The etiologic agent, the bacterium Tropheryma whipplei , cannot be cultivated in clinical laboratories, making the diagnosis of this culture-negative infection challenging. Molecular methods have emerged as useful adjuncts for the diagnosis of culture-negative endocarditis. Case Summary A 67-year-old male was seen in an infectious disease clinic for evaluation of a possible infectious etiology of chronic musculoskeletal pain with exercise intolerance. He had a history of an embolic stroke 2 years earlier, echocardiographic evidence of aortic valve thickening, and multiple negative blood cultures. Following an evaluation that included serology and extended incubation blood culture, plasma was sent for metagenomic sequencing of microbial cell-free DNA, which was positive for Tropheryma whipplei . Conclusion The patient’s musculoskeletal complaints and his exercise intolerance resolved after treatment with ceftriaxone and trimethoprim-sulfamethoxazole. To our knowledge, this is the first report of T. whipplei native valve endocarditis diagnosed by metagenomic sequencing.

Circulating Bacteriophage DNA Distinguishes Staphylococcal Infection From Commensal Colonization.

Comparative Analysis of TP53-Mutated Tumor DNA in Saliva and Plasma of Patients with Head and Neck Squamous Cell Carcinoma.

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive disease with a 5-year survival rate of 12%. Patients are typically diagnosed at the metastatic stage due to lack of symptoms and current treatment options do not effectively control the disease. There is an urgent need to develop sensitive strategies to detect PDA at an early stage before it spreads. Characterizing circulating tumor DNA (ctDNA) in plasma is an effective approach to detect and monitor cancer. Typically, cancer genomes contain thousands of mutations, but targeted sequencing approaches only focus on pre-determined driver genes, which limits their performance in detecting ctDNA. Whole-genome sequencing (WGS) however, tracks all the tumor mutations simultaneously, and has a higher sensitivity than targeted approaches, especially in low tumor burden samples. In this study, we aim to use plasma WGS to build a liquid biopsy platform for PDA detection and characterization. To date, we have established the largest plasma WGS (20-60x) PDAC cohort which consists of over 250 baseline and longitudinal plasma samples from 205 PDA patients, with an additional 45 plasma samples from age-matched healthy individuals. Paired tumor tissue WGS and RNAseq are also available. We developed and validated a bioinformatics pipeline to detect cancer mutations in plasma and estimate plasma tumor fraction (TFx) for tumor burden. In the PDA cohort, plasma TFx increased with clinical stages (medians of 0.5% in resectable, 0.8% in locally advanced and 5% in metastatic), as did the ctDNA detection rate which was measured by whether the plasma TFx was significantly higher than controls (87%, 91% and 95%). Plasma TFx positively correlated with liver metastasis size (R=0.68, P&amp;lt;0.001), but not with the primary pancreatic tumor size. These results suggest that in PDA, metastases are the major site to shed ctDNA, but not primary tumors. We also found that liver metastasis size had a stronger correlation with plasma TFx than with serum CA19-9 (R=0.32, P=0.002). Baseline plasma TFx was associated with shorter overall survival (OS) in both resectable (P=0.013) and metastatic (P=0.022) cases. By analyzing the paired tumor WGS and RNAseq, we found that higher plasma TFx was associated with higher cell cycle, lower immunity and polyploid genomes in the tumors. No difference was found in plasma TFx between basal-like and classical PDA subtypes. In summary, the current study provides insights into the interplay between ctDNA dynamics, tumor biology and clinical features in PDA. Alongside with these results, we will provide a freely accessible computational platform for ctDNA genomic profiling, PDA early detection and progression monitoring using plasma. Citation Format: Yuanchang Fang, Michelle Chan-Seng-Yue, Karen Ng, Amy Zhang, Tuan Hoang, Gun Ho Jang, Sabiq Chaudhary, Eugenia Flores-Figueroa, Daniela Bevacqua, Stephanie Ramotar, Ayelet Borgida, Shawn Hutchinson, Anna Dodd, Julie Wilson, Barbara Grünwald, Robert Grant, Erica Tsang, George Zogopoulos, Jennifer Knox, Masoom Haider, Steven Gallinger, Faiyaz Notta. Plasma whole-genome sequencing to monitor pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B072.

Association of psychosocial stress and poverty with plasma and extracellular vesicle mitochondrial DNA levels.

Non-thermal plasma has attracted strong interest in medicine and agriculture due to its ability to generate reactive oxygen and nitrogen species (RONS). These species can stimulate wound healing and seed germination, but at higher levels they induce DNA damage—useful in cancer therapy but harmful when healthy cells must be preserved. Direct study of DNA damage in cells is difficult because of repair processes and protective barriers. To address this, we applied a dual-model system combining plasmid DNA and human lymphocytes exposed to plasma from the RPS40 device. Using selective scavengers, we identified hydroxyl radicals, ozone, and reactive nitrogen species as key mediators of DNA strand breaks and structural changes. Our results support a mechanistic model in which long-lived plasma-derived species (NOx, ozone, acids) dissolve in water and subsequently generate short-lived radicals such as hydroxyl radicals and peroxynitrite. These reactive molecules then directly attack DNA. This integrated approach—linking plasmid and cellular assays with scavenger-based identification of RONS—offers a novel and cost-effective method for dissecting plasma–DNA interactions. The findings provide mechanistic insight into how plasma-activated water damages DNA, guiding the safer and more effective application of plasma technologies in biomedical and agricultural contexts.

Prostate cancer (PCa) is the second most common cancer and the fourth leading cause of cancer death in men worldwide. PSA screening for PCa diagnosis is not disease-specific; the discovery of novel and efficient biomarkers is therefore recommended. The concentration and integrity of circulating cell-free DNA (ccfDNA) in the blood of PCa patients could represent innovative and more specific tools for the clinical management of PCa. Digital droplet PCR (ddPCR) was used to determine the copy number ratio of ALU 260/111 bp and LINE-1 266/97 bp in the plasma of a cohort of 40 PCa and 18 BPH patients in a blinded prospective study. The amount of ccfDNA in the plasma of PCa and BPH patients was calculated from the EEF1A2 and ESR1 gene copy numbers. The ALU 260/111 and LINE-1 266/97 copy number ratios were significantly lower in the plasma of PCa patients compared to benign prostatic hyperplasia (BPH) ones (p-value; ALU 260/111: 0.006; LINE-1 266/97: 0.037). The area under the curve (AUC) indicated a good accuracy of two ratios and their product (ALU 260/111 * LINE 266/97, A*L) in discriminating PCa patients from BPH ones (AUC; ALU 260/111: 0.72; LINE-1 266/97: 0.67; A*L: 0.76). The ccfDNA concentration measured by EEF1A2 and ESR1 targets was significantly higher in the plasma of PCa patients compared to BPH patients, (p-value: EEF1A2, 0.017; ESR1, 0.024). The pilot ddPCR analysis of the ALU 260/111 and LINE-1 266/97 ratios in plasma indicates a new, reproducible and specific method for improving the early diagnosis of PCa. Further studies on larger cohorts are needed to confirm the results and clinical application.

Extracellular DNA of blood plasma (cell-free DNA, cfDNA) may potentially indicate a total level of apoptosis and mediate the immune response to stress induced by extreme environmental conditions, such as Antarctic wintering. We studied blood nuclease activity (NA); the content of 8-oxodG, rDNA, and SatIII(1q12) in cfDNA; and the levels of BAX, BCL2, TLR9, AIM2, STING, RIG-I, NF-kB, IL-8, and IL-17A mRNAs in 11 males, the members of the 64th Russian Antarctic Expedition. Blood was sampled before the wintering and on the 27th, 85th, 160th, 270th, and 315th days. The early months of the wintering are characterized by increased rates of apoptosis, an elevated BAX/BCL2 RNA ratio in blood leukocytes, and high cfDNA concentrations and NA in blood plasma. The properties of cfDNA are dramatically changed: the content of GC-rich rDNA rises, while AT-rich SatIII and 8-oxodG are low. We note individual multidirectional changes in the expression of TLR9 and AIM2, while STING and RIG-I are downregulated in all of the subjects. The mRNA levels of NFKB1, IL-8, and IL-17A increase dramatically, indicating immune system activation. In conclusion, (1) apoptosis is overactivated and remains elevated during the first half of the Antarctic wintering; (2) cfDNA is enriched with GC-repeats, which stimulates its biological activity; and (3) the expression of immunity genes associated with the inflammatory response is increased.

Longitudinal Circulating Tumor DNA-Guided Resistance Analysis During Second-Line Osimertinib Treatment.

Background: Recently, there has been a lot of interest in environmentally friendly nanoparticle synthesis. Silver nanoparticles (AgNPs) are promising against antibiotic resistance due to their high surface energy, robust action, and excellent adsorbability. Aim: The primary objective of this study was to assess the antibacterial efficacy of AgNPs that were manufactured using three environmentally friendly methods (lemon, Streptomyces, and chitosan). Furthermore, the study attempted to investigate the potential toxicity of these nanoparticles on mice. Methods: The synthesis of AgNPs was characterized by XRD, TEM FTIR, and TGA. The antimicrobial effect of AgNPs was studied using the disc diffusion method and minimum inhibitory concentration (MIC). The antibacterial mechanism of AgNPs was determined using different methods, such as released glucose and proteins, respiratory chain inhibition, plasma membrane fluorescence anisotropy, DNA fragmentation, gel electrophoresis, and cell membrane potential. Results: The TEM analysis of Ag NPs showed predominantly spherical particles with a size distribution of 10–60 nm. AgNPs synthesized by the three green methods showed antibacterial and fungal activity. The antibacterial mechanisms of AgNPs involved inhibition of LDH activity, increased protein and glucose leakage, DNA and protein damage, and depolarization and destabilization of the plasma membrane. AgNPs, on the other hand, increased alanine aminotransferase, aspartate aminotransferase, urea, creatinine, malondialdehyde, and nitric oxide levels in mice while decreasing glutathione reduced. Conclusion: Our study showed that AgNPs synthesized by Streptomyces, lemon, and chitosan have powerful antimicrobial properties. Chitosan-AgNPs showed the most pronounced antibacterial action, although it displayed significant toxicity in mice. Conversely, lemon-AgNPs revealed the least notable impact.

Target recycling amplification (TRA) combined with multiple strand displacement amplification (SDA) for sensitive detection of Epstein-Barr virus microRNA.

ABSTRACT Cellular senescence is a stable cell cycle arrest associated with upregulated inflammatory responses. Senescent cells contribute to various pathological and physiological processes including organismal ageing and cancer. Cellular senescence can be induced by various cellular stresses including DNA damage, telomere shortening, oncogene activation, and epigenetic alterations. We have shown that plasma membrane damage can also induce cellular senescence. However, common and specific molecular mechanisms among different senescent cell subtypes remain unknown. MicroRNAs (miRNAs) regulate mRNA and rewire gene expression profiles, contributing to multiple processes including cellular senescence. Here, we performed time-resolved miRNA sequencing and compared the results with mRNA sequencing results using cells experiencing plasma membrane damage-dependent senescence (PMD-Sen) and cells undergoing DNA damage response-dependent senescence (DDR-Sen). We found 65 miRNAs that are differentially regulated in PMD-Sen, contributing to 2,495 miRNA-mRNA pairs. Moreover, PMD-Sen and DDR-Sen shared 41 miRNAs across their sets of miRNA-mRNA pairs. Notably, miR-155-5p emerged as the miRNA with the largest number of shared miRNA-mRNA pairs that exhibit a highly negative correlation. These results highlight miR-155-5p as the potential key regulator of PMD-Sen and DDR-Sen.