Circulating cell-free DNA (ccfDNA) quantity correlates with the clinical characteristics and prognosis of various cancer types. We investigated whether ccfDNA levels and the neutrophil-to-lymphocyte ratio (NLR) have prognostic value in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral blood was collected from 82 patients with PDAC prior to any diagnostic procedure or the administration of chemotherapy. Plasma DNA was isolated, and ccfDNA concentration and NLR were determined. We found that ccfDNA levels were correlated with age and tumor burden. Moreover, higher values of NLR (≥3.31) were linked with worse overall survival (OS) (4 vs. 10 months; log rank p = 0.011), and an elevated ccfDNA concentration (≥25.79 ng/mL) was strongly associated with shorter OS (4 vs. 8 months; log rank p = 0.009). According to the results of the multivariable Cox regression analysis, the baseline concentration of ccfDNA was an independent prognostic factor for OS (HR 0.45, 95% CI 0.21–0.97, p = 0.041). Furthermore, the combination of ccfDNA levels with NLR greatly enhanced the prognostic accuracy of PDAC patients. Our study demonstrates that ccfDNA concentration and NLR are independent predictors of survival in PDAC. Subsequent studies should validate this combination as a prognostic indicator in PDAC patients and assess its utility for guiding therapeutic decisions.

Background: Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers worldwide. Alpha-fetoprotein (AFP), a widely used and accessible tumoral marker, has limited performance in the early detection of HCC among high-risk populations. This study aims to evaluate the potential added value of ccfDNA (circulating cell-free DNA) fragment size, alone or in a multiparameter panel, using accessible, feasible ccfDNA analysis. Methods: A prospective cohort of 125 patients with chronic liver disease was analyzed. Patients with incomplete clinical or laboratory data and patients without cirrhosis were excluded from the final analysis. Nonparametric tests, logistic regression and ROC curve analysis were performed. ccfDNA fragment size was measured using on-chip electrophoresis. Results: ccfDNA fragment size was significantly lower in the cirrhosis-HCC subgroup compared to the cirrhosis-only subgroup (p < 0.001). While AFP remains an independent predictor of HCC among cirrhosis patients, ccfDNA fragment size did not prove to be an independent predictor in this cohort. AUROC (area under the receiver operating characteristic curve) analysis revealed that a combined model of AFP, age, liver reserve, and ccfDNA fragment size did not perform better than the corresponding panel without ccfDNA. Moreover, after DeLong comparison, the difference between the two AUROCs proved statistically insignificant. Age and platelet count remain the strongest independent predictors in our exploratory cohort. Conclusions: Although ccfDNA fragment size proved to be lower in the HCC subgroup, its statistical significance fades when included into a multimarker panel. However, all panels should undergo further validation in a larger cohort, in order to better assess the individual contribution of each parameter and to discriminate between added diagnostic value and confounding effect of age and liver reserve parameters.

Background: Chemokine CCL5 may drive inflammation and vascular risk in advanced liver disease, but its cardiovascular implications are unclear. Secreted by hepatic, endothelial, macrophage, and lymphocytic cells, CCL5 is involved in cytokine regulation. Its serum levels rise in acute liver injury and hepatocellular carcinoma (HCC), but decline with fibrosis progression in end-stage liver disease (ESLD). CCL5 has also been linked to atherosclerosis. This study aimed to evaluate serum CCL5 levels in ESLD patients listed for liver transplantation (LT) and to assess their potential role as markers of cardiovascular (CV) risk and portal hypertension. Methods: We conducted an observational cohort study. Between 2019 and 2022, patients with ESLD evaluated for LT were enrolled. Data on liver pathology, CV risk, and laboratory parameters were collected. Serum CCL5 concentrations were measured using Sigma Aldrich^® CCL5 ELISA kits (MilliporeSigma, St. Louis, MO, USA). The database was analyzed with IBM^® SPSS^® Statistics version 20 (Chicago, IL, USA). Results: Overall, 46 patients were included, 50% with viral hepatitis and 28.3% with alcohol-related liver disease. HCC was present in 37% of cases. The median CV risk scores (CAD_LT = 7, mCAD_LT = 7, CAR_OLT = 18) placed the population at moderate CV risk. Serum CCL5 levels did not vary significantly between viral vs. non-viral cirrhosis (5511.8 vs. 6272.5 pg/mL, p = 0.15) and were not influenced by the presence of HCC (6098.4 vs. 5771.3 pg/mL, p = 0.55). We did not detect a correlation with MELD score (p = 0.21) or CV risk scores (CAD_LT: p = 0.58; mCAD_LT: p = 0.70; CAR_OLT: p = 0.22). Patients with thrombocytopenia (<100,000/µL, 54.3%) or a history of esophageal variceal ligation had lower CCL5 levels (5170.9 vs. 6750.8 pg/mL, p = 0.002 and 4252.0 vs. 6237.5 pg/mL, p = 0.003, respectively). Similarly, patients with a history of previous variceal bleeding and spontaneous bacterial peritonitis (SBP) had lower levels of CCL5 (4373.8 vs. 6119.9 pg/mL, p = 0.02 and 3404.3 vs. 6606.7 pg/mL, p = 0.01, respectively). We found a negative correlation between CCL5 and QTc interval duration (τ = −0.216, p = 0.037), left ventricle size (LV: τ = −0.235, p = 0.027), and pulmonary artery pressure (RV/RA gradient: τ = −0.225, p = 0.03). CCL5 correlated positively with the inflammatory markers C-reactive protein (CRP) (τ = 0.246, p = 0.018) and fibrinogen (r = 0.216, p = 0.04). Conclusions: In liver transplant candidates, serum CCL5 is not associated with cardiovascular risk scores or coronary atherosclerotic burden, but is inversely associated with clinical markers of portal hypertension severity. These findings suggest that CCL5 may serve as a potential non-invasive surrogate marker of portal hypertension rather than a cardiovascular risk biomarker in ESLD.

Background/Objectives: Given the ongoing threat of antimicrobial resistance, the identification and characterization of multidrug-resistant isolates are essential. An increase in antimicrobial-resistant bacteria has been reported in Romania, but national data are still scarce. This study aimed to evaluate beta-lactamase-producing Gram-negative bacteria (GNB) isolated over two years at a Romanian nephrology hospital, while comparing carbapenemase detection phenotypic methods. Methods: Gram-negative bacterial isolates collected between January 2022 and May 2024 that met antimicrobial resistance screening criteria were evaluated. After identification, extensive disk diffusion antibiograms were performed, read, and interpreted, complemented by testing on cloxacillin/oxacillin-supplemented Mueller–Hinton agar. The colistin minimum inhibitory concentration (MIC) was not assessed, and aztreonam–avibactam was not tested for Enterobacterales. For non-fermenter GNB, the colistin MIC was determined. Phenotypic carbapenemase production tests were performed for all strains (BlueCarba Test, CIM, mCIM, zCIM, and rCIM). Carbapenemase detection immunochromatographic tests were performed for a set of strains. Results: Among the 397 evaluated strains, 335 (84.38%) were Enterobacterales and 62 (15.62%) non-fermenter GNB, showing high antimicrobial resistance levels. Of these, 188 (47.35%) were Klebsiella pneumoniae; 139/188 (73.93%) showed carbapenem resistance and carbapenemase production; 49/188 (26.06%) produced two carbapenemases; and 45/188 (23.93%) presented resistance to all tested antimicrobials. MALDI-TOF identified 28 KPC-producing K. pneumoniae strains. Lateral flow assays revealed NDM, VIM, KPC, and OXA-48-like enzymes in 48 of 56 tested Enterobacterales; 12/48 strains produced two carbapenemases. Of the 62 non-fermenter GNB, 33 were Pseudomonas spp. and 20 Acinetobacter baumannii; one Pseudomonas spp. was susceptible only to colistin and seven only to cefiderocol; four A. baumannii were susceptible only to colistin and three only to cefiderocol. Lateral flow assays detected VIM or IMP enzymes in 13/33 Pseudomonas spp. and OXA-23 and/or OXA-40/-58 enzymes in all 20 A. baumannii. Conclusions: Among the evaluated strains, many showed resistance to multiple antimicrobial classes. Furthermore, strains co-producing two carbapenemases were identified.

Introduction: Circulating cell-free DNA (cfDNA) has emerged as a promising minimally invasive biomarker in hepatocellular carcinoma (HCC), with potential applications in disease detection and prognostic stratification. This exploratory study aimed to evaluate the relationship between circulating cell-free DNA (cfDNA) concentration, liver dysfunction parameters, and hepatocellular carcinoma stage in an exploratory cohort: we sought to explore the extent to which cfDNA variability may be explained by the underlying liver disease environment and whether cfDNA concentration provides incremental information beyond routinely available markers of liver reserve for the discrimination between early- and late-stage hepatocellular carcinoma. Methods: Sixty-four newly diagnosed HCC patients were included. Clinical, laboratory, and staging data were collected. cfDNA was isolated from plasma, confirmed by on-chip electrophoresis, and quantified by fluorimetry. Logistic regression and ROC curve analyses were performed to assess the ability of several biomarker panels to discriminate early-stage HCC (BCLC 0–A) from intermediate/advanced-stage disease (BCLC B–D). Bootstrap resampling (2000 iterations) evaluated model robustness and coefficient stability. Additional linear regression analyses explored associations between cfDNA concentration and liver dysfunction parameters. Results: Linear regression demonstrated that liver dysfunction parameters explained approximately 53% of cfDNA variability, while HCC stage contributed minimally after adjustment. Models incorporating albumin, bilirubin, and platelet count as individual parameters demonstrated the best discriminatory performance after adjustment for liver disease etiology, achieving AUROCs up to 0.857. The only incremental value that cfDNA concentration added to the panel was an increase to its specificity (from 79.4% to 94.1%), while reducing sensitivity by 13.4%. Bilirubin and platelet count remained the most stable predictors after bootstrap, whereas cfDNA concentration was unstable. Limitations: The study was limited by its small sample size, cross-sectional design, and lack of longitudinal outcome assessment. External validation in larger prospective cohorts is necessary. Conclusion: A substantial proportion of cfDNA concentration variability (approximately 53%) was explained by routinely available liver dysfunction parameters, whereas HCC stage had minimal contribution. These findings suggest that circulating cfDNA concentration in patients with HCC may be influenced to a greater extent by the underlying cirrhotic liver environment and hepatocyte injury than by tumor burden alone. Integrated multimarker panels combining liver reserve parameters and liver disease etiology may be of interest for minimally invasive stratification of HCC. Although cfDNA concentration increased specificity for early- versus advanced-stage disease discrimination, its incremental value was low. Further validation in larger prospective cohorts is required.

Background: Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers worldwide. Alpha-fetoprotein (AFP), a widely used and accessible tumoral marker, has limited performance in the early detection of HCC among high-risk populations. This study aims to evaluate the potential added value of ccfDNA (circulating cell-free DNA), alone or in combination with AFP, using accessible, feasible ccfDNA analysis. Methods: A prospective cohort of 125 patients with chronic liver disease was analyzed. Patients with incomplete clinical or laboratory data and patients without cirrhosis were excluded from the final analysis. Nonparametric tests, logistic regression and ROC curve analysis were performed. ccfDNA concentration was assessed by fluorimetry and fragment size was measured using on-chip electrophoresis. Results: ccfDNA fragment size was significantly lower in the cirrhosis-HCC subgroup compared to the cirrhosis-only subgroup (p< 0.001). While AFP remains an independent predictor of HCC among cirrhosis patients, ccfDNA fragment size did not prove to be an independent predictor in this cohort. However, AUROC (Area Under the Receiver Operating Characteristic Curve) analysis revealed that a combined model of AFP and ccfDNA fragment size showed modest additional discriminatory value between the two groups, compared to either ccfDNA peak size or AFP alone. Conclusions: ccfDNA fragment size may provide modest complementary value within multimarker panels. However, the marker needs further validation in a larger cohort, and adequate assessment of potential confounders such as severity of liver dysfunction and age.