Cell-free Deoxyribonucleic Acid Research Articles

Culture time to optimize embryo cell-free DNA analysis for frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy

To investigate the ideal time in culture to optimize embryo cell-free deoxyribonucleic acid (cfDNA) analysis in frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy (PGT-A). Cell-free DNA is released into the spent blastocyst media (spent media) by the embryo. However, the optimal timing to determine maximal cfDNA in the case of frozen-thawed blastocysts undergoing noninvasive PGT-A remains to be elucidated. In this prospective observational study, 135 spent media and corresponding whole blastocysts were collected from January 2021 through March2022. Private fertility clinics. Day-5 frozen-thawed blastocysts were cultured for 8 hours (Day-5 Short) or 24 hours (Day-5 Long), whereas day-6 frozen-thawed blastocysts were cultured for 8 hours (Day-6 Short). The spent media and whole blastocysts were then collected for further analysis. Spent media and whole blastocysts were amplified using whole genome amplification and sequenced using next-generation sequencing. Informativity and concordance rates between cfDNA in spent media and whole blastocyst DNA were compared according to the different times in culture. When comparing time in culture, informativity rates for spent media were significantly higher for Day-5 Long and Day-6 Short (>91%) compared with the Day-5 Short group (<60%). A similar trend was observed for cases with and without a previous PGT-A. Regarding blastocyst expansion grade, informativity rates were lower on Day-5 Short compared with Day-5 Long and Day-6 Short, regardless of expansion degree. This decrease was significant for Gardner-grade expansion grades 3, 4, and 5-6. In addition, for a similar time in culture, the grade of expansion did not have an impact on the informativity rates. For concordance rates, no significant differences were observed among the 3 groups. In all cases, concordance rates were 90.5% for Day-5 Short, 93.6% for Day-5 Long, and 92.3% for Day-6 Short. No impact of the expansion grade was observed on concordance rates. Noninvasive PGT-A in frozen-thawed blastocysts yields very high concordance rates with whole blastocysts, possibly limiting the need for invasive PGT-A and making it available for a wider range of patients.

Damage-Associated Molecular Patterns as Mediators of Thrombus Formation on Dialyzer Membrane in Critically Ill Patients.

This prospective study investigated the relationship between inflammation, damage-associated molecular patterns (DAMPs), and thrombus formation on dialyzer membranes in critically ill patients undergoing renal replacement therapy (RRT) from July 2020 to August 2022, identifying mechanisms and interventions to prevent clotting. The patients were divided into two groups: inflammatory (n = 56, serum C-reactive protein >10 mg/dl) and noninflammatory control (n = 45, serum C-reactive protein <5 mg/dl). Cell-free deoxyribonucleic acid (DNA) levels, high mobility group box 1 protein (HMGB1), histone H3, and myeloperoxidase (MPO) in the lumen of the hollow fiber membrane of the dialyzer were quantified. Immunostaining assessed leukocytes, fibrin fibers, and platelet thrombi on the luminal surface of the hollow fiber membrane. The inflammatory group, compared to controls, exhibited elevated cell-free DNA, HMGB1, and MPO levels, although histone H3 remained unchanged. Damage-associated molecular patterns increased with disseminated intravascular coagulation (DIC) severity. Immunostaining in the inflammatory group revealed leukocytes, amorphous nuclei, neutrophil extracellular trap-like structures, fibrin fibers, and platelet thrombi on the hollow fiber membrane's luminal surface. Elevated DAMP levels in severely inflamed patients' dialyzer membranes, correlating with DIC severity, indicate a link between inflammation, coagulation activation, and dialyzer clotting. Research into thrombus prevention in RRT for DIC-affected critically ill patients is warranted.

Neutrophil Extracellular Trap Formation and Deoxyribonuclease I Activity in Patients with Otitis Media with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

Introduction: No previous studies have evaluated the levels of neutrophil extracellular trap (NET) remnants or the importance of deoxyribonuclease (DNase) I activity based on the disease activity of otitis media with antineutrophil cytoplasmic antibody-associated vasculitis (OMAAV). The aim of this study was to explore the formation of NETs in the middle ear of patients with OMAAV during the onset and remission phases of the disease, with a particular focus on the relationships between the quantifiable levels of NET remnants and DNase I activity. Methods: OMAAV patients were eligible for inclusion. Patients with otitis media with effusion (OME) were examined as controls. The levels of cell-free deoxyribonucleic acid (DNA), citrullinated-histone H3 (cit-H3)-DNA complex, and myeloperoxidase (MPO)-DNA complex were quantified using an enzyme-linked immunosorbent assay. DNase I activity was measured using a fluorometric method. Results: The quantifiable levels of cell-free DNA, cit-H3-DNA complex, and MPO-DNA complex in the middle ear lavage of patients with OMAAV at onset were significantly higher than those in patients with OMAAV at remission and in patients with OME. DNase I activity in the patients with OMAAV at onset was significantly lower than those in patients with OMAAV at remission and OME and was negatively correlated with the level of MPO-DNA complex. Conclusions: This study suggests that NET remnants and DNase I activity may be potentially useful biomarkers for the diagnosis and disease activity of OMAAV.

Fragmentation patterns of cell-free DNA and somatic mutations in the urine of metastatic breast cancer patients.

Urinary cell-free deoxyribonucleic acid (DNA) (ucfDNA) holds promise as a biomarker; however, its potential remains largely unexplored. We examined the fragmentation pattern of ucfDNA and identified somatic mutations within urine samples from metastatic breast cancer (MBC) patients. Urine and blood specimens were collected before treatment from 45 MBC patients and posttreatment urine samples from 16 of the 45 patients at the China National Cancer Center. Somatic mutations and tumor mutational burden (TMB) in the urine and plasma of 10 patients were analyzed by next-generation sequencing (NGS). Fragmentation patterns of cfDNA were displayed using electropherograms. Differences in the extracted amount of cfDNA, length of cfDNA fragments, and TMB between urine and plasma were compared using a Wilcoxon test. The fragmentation patterns of ucfDNA were categorized as follows: (1) profile A (n = 26) containing a short peak (100-200 bp) and a long peak (>1500 bp); (2) profile B (n = 8) containing only a long peak; and (3) profile C (n = 11) containing flat pattern. For profile A patients, the short-peaked ucfDNA circulating in the bloodstream was much shorter compared with plasma cfDNA (149 vs. 171 bp, Wilcoxon test, P = 0.023). The fragmentation patterns in lung metastasis patients exhibited a higher propensity toward profile C ( P = 0.002). After treatment, 87.5% of the patients exhibited consistent fragmentation patterns. The concordance rate for somatic mutations in the plasma and urine was 30%, and the median TMB of urine and plasma was not significantly different. This study established a fragmentation pattern for ucfDNA and detected somatic mutations in the urine of MBC patients. These results suggest the potential application of ucfDNA as a biomarker for MBC.

The emerging role of cell-free DNA as a molecular marker for duodenal adenocarcinoma.

Duodenal adenocarcinoma is rare and its prognostic factors remain controversial. In our study, the role of cell-free deoxyribonucleic acid (cfDNA) as prognostic factor in duodenal adenocarcinoma was evaluated. From June 2003 to July 2021, plasma samples were collected from 41 patients with duodenal adenocarcinoma. Plasma cfDNA was assessed in combination with clinicopathological and biochemical characteristics. Univariate and multivariate analyses were conducted to identify independent prognostic factors for overall survival with a Cox proportional hazards regression model. The 1- and 5-year survival rates of the patients with high plasma cfDNA level (>9288 copies/mL) group were 58.7% and 17.6%, respectively, which were much lower than patients with low cfDNA level (≤9288 copies/mL), with 95.2% and 64.6%. In univariate analysis, high cfDNA level, lymph node involvement, lymphovascular invasion, and tumor stage were associated with decreased survival. When subjected to multivariate analysis, only high cfDNA level showed significance in influencing the overall survival of duodenal cancer. cfDNA analysis is simple and noninvasive. High cfDNA level is a strong independent prognostic factor for decreased overall survival and it should be integrated into clinical care.

A case report of carcinoma of the papilla of Vater associated with a hyperplasia-dysplasia-carcinoma sequence by pancreaticobiliary maljunction.

Pancreaticobiliary maljunction (PBM) is a known risk factor for biliary tract cancer. However, its association with carcinoma of the papilla of Vater (PVca) remains unknown. We report a case with PVca that was thought to be caused by the hyperplasia-dysplasia-carcinoma sequence, which is considered a mechanism underlying PBM-induced biliary tract cancer. A 70-year-old woman presented with white stool and had a history of cholecystectomy for the diagnosis of a non-dilated biliary tract with PBM. Esophagogastroduodenoscopy revealed a tumor in the papilla of Vater, and PVca was histologically proven by biopsy. We finally diagnosed her with PVca concurrent with non-biliary dilated PBM (cT1aN0M0, cStage IA, according to the Union for International Cancer Control, 8th edition), and subsequently performed subtotal stomach-preserving pancreaticoduodenectomy. Pathological findings of the resected specimen revealed no adenomas and dysplastic and hyperplastic mucosae in the common channel slightly upstream of the main tumor, suggesting a PBM related carcinogenic pathway with hyperplasia-dysplasia-carcinoma sequence. Immunostaining revealed positivity for CEA. CK7 positivity, CK20 negativity, and MUC2 negativity indicated that this PVca was of the pancreatobiliary type. Genetic mutations were exclusively detected in tumors and not in normal tissues, and bile ducts from formalin-fixed paraffin-embedded samples included mutated-ERBB2 (Mutant allele frequency, 81.95%). Moreover, of the cell-free deoxyribonucleic acid (cfDNA) extracted from liquid biopsy mutated-ERBB2 was considered the circulating-tumor deoxyribonucleic acid (ctDNA) of this tumor. Herein, we report the first case of PVca with PBM potentially caused by a "hyperplasia-dysplasia-carcinoma sequence" detected using immunostaining and next-generation sequencing. Careful follow-up is required if pancreaticobiliary reflux persists, considering the possible development of PVca.

A pilot study to investigate the clinically predictive values of copy number variations detected by next-generation sequencing of cell-free deoxyribonucleic acid in spent culture media

To investigate the positive predictive value and false positive risk of copy number variations (CNV's) detected in cell free deoxyribonucleic acid (DNA) from spent culture media for nonviable or aneuploid embryos. Diagnostic/prognostic accuracy study. Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF center. Embryo selection was performed according to the conventional grading, blinded to noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) results. After clinical outcomes were established, spent culture media samples were analyzed. Prognostic accuracy of CNVs according to niPGT-A results to predict nonviability or clinical aneuploidy. One hundred twenty patients completed the study. Interpretations of next-generation sequencing (NGS) profiles were as follows: 7.5% (n = 9) failed quality control; 62.5% (n = 75) no CNVs detected; and 30% (n = 36) abnormal copy number detected. Stratification of abnormal NGS profiles was as follows: 15% (n = 18) whole chromosome and 15% (n = 18) uncertain reproductive potential. An intermediate CNV was evident in 27.8% (n = 5) of the whole chromosome abnormalities. The negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk (RR) for nonviability compared with no detected abnormalities (RR 2.21, 95% CI: 1.66-2.94). No other CNVs were associated with significant differences in the sustained implantation or RRs for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant median absolute pairwise differences between certain subcategories of CNV abnormalities were apparent. Whole chromosome abnormalities were associated with a high positive predictive value and significant RR for nonviability. Embryos associated with other CNVs had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of niPGT-A. clinicaltrials.gov (NCT04732013).

Is there a role for liquid biopsy in the surveillance of parathyroid carcinoma?

BackgroundParathyroid carcinoma is a rare malignancy with high recurrence rates. Liquid biopsy is a stratifying tool in disease recurrence/progression in other malignant processes. This study sought to assess the feasibility and application of liquid biopsy in parathyroid carcinoma and its impact on surveillance. MethodsRetrospective review of a prospectively maintained database of adults treated for parathyroid carcinoma at a tertiary care center (2017–2023). Demographics, clinical characteristics, and laboratory variables were collected. Circulating cell-free deoxyribonucleic acid enrichment and circulating tumor cell enumeration were obtained from serial blood samples. ResultsA total of 25 patients were identified—64% were male patients, with a median age of 56 years (interquartile range 45–63). Fifty blood samples were collected postoperatively. At first, circulating tumor cell enumeration, 56% (14/25) of patients had no evidence of disease, and 32% (8/25) had distant metastasis. Median follow-up was 53 months (interquartile range 23–107). At the last follow-up, 40% (10/25) of patients were found to have distant metastasis. Serial circulating tumor cell enumeration was performed in 52% of patients, median highest circulating tumor cell was (interquartile range 1–22). Circulating cell-free deoxyribonucleic acid was assessed in 64% of patients (16/25). There was no difference in circulating tumor cells or circulating cell-free deoxyribonucleic acid between those with distant metastasis and those without distant metastasis. The most common mutation identified was TP53, present in 88% of circulating cell-free deoxyribonucleic acid samples with a mutation. Circulating cell-free deoxyribonucleic acid and parathyroid hormone levels were not found to have any association (r = −0.27, P = .39), but parathyroid hormone and circulating tumor cell had a linear relationship (r = 0.76, P < .001). ConclusionLiquid biopsy appears to be a feasible tool in parathyroid carcinoma surveillance. The relationship between circulating cell-free deoxyribonucleic acid and parathyroid hormone levels remains unclear, and the association between circulating tumor cell enumeration and parathyroid hormone levels may be impactful. The finding that TP53 mutation is more prevalent in patients with distant metastasis may impact further management.

Impact of cell-free DNA fungal polymerase chain reaction panels on healthcare-associated infection mould investigations.

Launch of in-house sensitive cell-free deoxyribonucleic acid (cfDNA) mould polymerase chain reaction (PCR) assays increased detection of moulds meeting suspected healthcare-associated infection (HAI) criteria. Definition was based on time from admission and mould detection in culture or via molecular methods. We created a modified mould HAI algorithm incorporating clinical context into the case definition, which allowed for better capture of possible mould HAIs, decreased number of investigations, and improved utilization of Infection Prevention and Control (IPC) resources.

Psychological Impact of the Galleri test (sIG(n)al): protocol for a longitudinal evaluation of the psychological impact of receiving a cancer signal in the NHS-Galleri trial

IntroductionMulti-cancer early detection (MCED) blood tests look for cancer signals in cell-free deoxyribonucleic acid. These tests have the potential to detect cancers at an earlier (asymptomatic) stage, improving cancer outcomes....

Infective Endocarditis during Pregnancy-Keep It Safe and Simple!

The diagnosis of infective endocarditis (IE) during pregnancy is accompanied by a poor prognosis for both mother and fetus in the absence of prompt management by multidisciplinary teams. We searched the electronic databases of PubMed, MEDLINE and EMBASE for clinical studies addressing the management of infective endocarditis during pregnancy, with the aim of realizing a literature review ranging from risk factors to diagnostic investigations to optimal therapeutic management for mother and fetus alike. The presence of previous cardiovascular pathologies such as rheumatic heart disease, congenital heart disease, prosthetic valves, hemodialysis, intravenous catheters or immunosuppression are the main risk factors predisposing patients to IE during pregnancy. The identification of modern risk factors such as intracardiac devices and intravenous drug administration as well as genetic diagnostic methods such as cell-free deoxyribonucleic acid (DNA) next-generation sequencing require that these cases be addressed in multidisciplinary teams. Guiding treatment to eradicate infection and protect the fetus simultaneously creates challenges for cardiologists and gynecologists alike.

Noninvasive prenatal screening and maternal malignancy: role of imaging.

Noninvasive prenatal screening (NIPS) tests for fetal chromosomal anomalies through maternal blood sampling. It is becoming widely available and standard of care for pregnant women in many countries. It is performed in the first trimester of pregnancy, usually between 9 and 12weeks. Fragments of fetal cell-free deoxyribonucleic acid (DNA) floating in maternal plasma are detected and analyzed by this test to assess for chromosomal aberrations. Similarly, maternal tumor-derived cell-free DNA (ctDNA) released from the tumor cells also circulates in the plasma. Hence, the presence of genomic anomalies originating from maternal tumor-derived DNA may be detected on the NIPS-based fetal risk assessment in pregnant patients. Presence of multiple aneuploidies or autosomal monosomies are the most commonly reported NIPS abnormalities detected with occult maternal malignancies. When such results are received, the search for an occult maternal malignancy begins, in which imaging plays a crucial role. The most commonly detected malignancies via NIPS are leukemia, lymphoma, breast and colon cancers. Ultrasound is a reasonable radiation-free modality for imaging during pregnancy, specially when there are localizing symptoms or findings, such as palpable lumps. While there are no consensus guidelines on the imaging evaluation for these patients, when there are no localizing symptoms or clinically palpable findings, whole body MRI is recommended as the radiation-free modality of choice to search for an occult malignancy. Based on clinical symptoms, practice patterns, and available resources, breast ultrasound, chest radiographs, and targeted ultrasound evaluations can also be performed initially or as a follow-up for MRI findings. CT is reserved for exceptional circumstances due to its higher radiation dose. This article intends to increase awareness of this rare but stressful clinical scenario and guide imaging evaluation for occult malignancy detected via NIPS during pregnancy.

Cell-free DNA in Embryo Culture Media as Non-invasive Biomarker of the Quality of Embryo Cleavage

Background: The success of in vitro-fertilization (IVF) cycles is determined in large part by the quality of embryo cleavage, which in turn, is dependent on the quality of the embryo culture media (CM). Many factors can influence the quality of embryo CM, one of which is the levels of Cell Free Deoxyribonucleic acid (DNA). Understanding the association between Cell-free DNA levels in embryo CM and the quality of embryo cleavage could help improve the quality of IVF techniques.&#x0D; Methods: This prospective study was conducted with 96 spent CM from patients undergoing IVF cycle, in order to determine relationships of Cell-free DNA levels in embryo CM with embryo cleavage quality on day 3. After intracytoplasmic sperm injection (ICSI), 48 embryos were evaluated on day 3 of their development, according to their cell number. Day 2 and day 3 CM corresponding to each one of the embryos was analyzed, by quantitative PCR, for estimation of Cell-free DNA levels.&#x0D; Results: The results revealed a significant increase in Cell-free DNA levels on day 2 CM corresponding to 4 to 6 cell embryos compared to those corresponding to 7 to 8 cell embryos (p=0.04). As for day 3 CM, the results showed no significant difference between the Cell-Free DNA levels in CM of 7-8 and those of 4-6 cell embryos (p=0.4). Also, cell free DNA levels in embryo CM, were significantly higher on day 2 compared to day 3 for both 7-8 and 4-6 cell embryos (p=0.03; p=0.04). &#x0D; Conclusion: We conclude that cell-free DNA levels in CM might be associated with delayed embryo cleavage.

IMperm: a fast and comprehensive IMmune Paired-End Reads Merger for sequencing data.

The adaptive immune receptor repertoire (AIRR), consisting of T- and B-cell receptors, is the core component of the immune system. The AIRR sequencing is commonly used in cancer immunotherapy and minimal residual disease (MRD) detection of leukemia and lymphoma. The AIRR is captured by primers and sequenced to yield paired-end (PE) reads. The PE reads could be merged into one sequence by the overlapped region between them. However, the wide range of AIRR data raises the difficulty, so a special tool is required. We developed a software package for IMmune PE reads merger of sequencing data, named IMperm. We used the k-mer-and-vote strategy to pin down the overlapped region rapidly. IMperm could handle all types of PE reads, eliminate adapter contamination and successfully merge low-quality and minor/non-overlapping reads. Compared with existing tools, IMperm performed better in both simulated and sequencing data. Notably, IMperm was well suited to processing the data of MRD detection in leukemia and lymphoma and detected 19 novel MRD clones in 14 patients with leukemia from previously published data. Additionally, IMperm can handle PE reads from other sources, and we demonstrated its effectiveness on two genomic and one cell-free deoxyribonucleic acid datasets. IMperm is implemented in the C programming language and consumes little runtime and memory. It is freely available at https://github.com/zhangwei2015/IMperm.

Cell-free deoxyribonucleic acid analysis in preimplantation genetic testing

Detection of chromosomal aneuploidies and monogenic disorders in preimplantation embryos is essential for selecting the best embryo for transfer during invitro fertilization to achieve a healthy pregnancy. Preimplantation genetic testing (PGT) is typically performed on preimplantation embryos to select a genetically normal embryo for transfer. A trophectoderm biopsy is necessary for PGT; this is an invasive procedure to the embryo that requires specialized equipment and highly trained embryologists, resulting in high costs associated with invitro fertilization treatment. Moreover, the biopsy procedure may increase the likelihood of developing pregnancy complications, such as preeclampsia and hypertensive disorders. Therefore, there is a need for noninvasive embryo screening strategies. The presence of cell-free deoxyribonucleic acid in the embryo culture medium presents an opportunity to screen for genetic abnormalities. Cell-free deoxyribonucleic acid is released by embryos in the latter stages of preimplantation development, and its analysis has been proposed as a noninvasive approach for PGT. Here, we review studies reporting the concordance rates between cell-free deoxyribonucleic acid and trophectoderm biopsies, or whole blastocysts, in couples undergoing PGT. Noninvasive PGT results are promising for aneuploidy detection, with some early evidence of successful clinical application. Further research is required to explore its application for the detection of structural rearrangements and monogenic disorders.

Neutrophil Extracellular Traps and Their Possible Implications in Ocular Herpes Infection.

Neutrophil extracellular traps (NETs) are net-like structures released from neutrophils. NETs predominantly contain cell-free deoxyribonucleic acid (DNA) decorated with histones and neutrophil granule proteins. Numerous extrinsic and intrinsic stimuli can induce the formation of NETs such as pathogens, cytokines, immune complexes, microcrystals, antibodies, and other physiological stimuli. The mechanism of NETosis induction can either be ROS-dependent or independent based on the catalase producing activity of the pathogen. NADPH is the source of ROS production, which in turn depends on the upregulation of Ca2+ production in the cytoplasm. ROS-independent induction of NETosis is regulated through toll-like receptors (TLRs). Besides capturing and eliminating pathogens, NETs also aggravate the inflammatory response and thus act as a double-edged sword. Currently, there are growing reports of NETosis induction during bacterial and fungal ocular infections leading to different pathologies, but there is no direct report suggesting its role during herpes simplex virus (HSV) infection. There are innumerable independent reports showing that the major effectors of NETosis are also directly affected by HSV infection, and thus, there is a strong possibility that HSV interacts with these facilitators that can either result in virally mediated modulation of NETosis or NETosis-mediated suppression of ocular HSV infection. This review focuses on the mechanism of NETs formation during different ocular pathologies, with its prime focus on highlighting their potential implications during HSV ocular infections and acting as prospective targets for the treatment of ocular diseases.

Liquidhope: methylome and genomic profiling from very limited quantities of plasma-derived DNA.

Analysis of the methylome of tumor cell-free deoxyribonucleic acid (DNA; cfDNA) has emerged as a powerful non-invasive technique for cancer subtyping and prognosis. However, its application is frequently hampered by the quality and total cfDNA yield. Here, we demonstrate the feasibility of very low-input cfDNA for whole-methylome and copy-number profiling studies using enzymatic conversion of unmethylated cysteines [enzymatic methyl-seq (EM-seq)] to better preserve DNA integrity. We created a model for predicting genomic subtyping and prognosis with high accuracy. We validated our tool by comparing whole-genome CpG sequencing with in situ cohorts generated with bisulfite conversion and array hybridization, demonstrating that, despite the different techniques and sample origins, information on cfDNA methylation is comparable with in situ cohorts. Our findings support use of liquid biopsy followed by EM-seq to assess methylome of cancer patients, enabling validation in external cohorts. This advance is particularly relevant for rare cancers like neuroblastomas where liquid-biopsy volume is restricted by ethical regulations in pediatric patients.

Nanogels designed for cell-free nucleic acid sequestration.

Chronic wounds exhibit over-expression of cell-free deoxyribonucleic acid (cfDNA), leading to a prolonged inflammation and non-healing wounds. Scavenging excessive cfDNA molecules is a promising strategy for chronic wound treatment. Nanoscopic particles act as efficient cfDNA scavengers due to their large surface area, however their efficiency in cfDNA uptake was limited by adsorption solely on the nanoparticle surface. In contrast, nanogels may provide multiple cfDNA binding sites in the nanoparticle interior, however their use for cfDNA scavenging is yet to be explored. Herein, we report cationic nanogels derived from a copolymer of chitosan and poly{2-[(acryloyloxy)ethyl]trimethylammonium chloride} end-grafted to the chitosan backbone as side chains. The nanogels retain their positive charge at the pH and ionic strength of chronic wound exudate, enabling electrostatically driven cfDNA scavenging. The network structure of the nanogels leads to the cfDNA sequestration in the nanogel interior, in addition to surface attachment. A key factor in cfDNA sequestration is the ratio of the pore size of the nanogel-to-cfDNA molecular dimensions. The enhanced cfDNA scavenging efficiency, along with biocompatibility of the nanogels, makes them a promising component of dressings for chronic wound treatment.

85. Plasma Cell Free Deoxyribonucleic Acid (cfDNA) Fungal Polymerase Chain Reaction (PCR): a Non-Invasive Diagnostic for Invasive Fungal Disease Evaluation in At-Risk Pediatric Oncology and Stem Cell Transplant Patients

Abstract Background Molecular diagnostics appear promising for early, non-invasive detection of invasive fungal disease (IFD) in immunocompromised patients. Our clinical lab developed and validated cell free DNA (cfDNA) fungal polymerase chain reaction (PCR) assays (Table 1), which have been in clinical use since November 2020 and were recently included in an institutional pediatric clinical care pathway for prolonged febrile neutropenia. We aimed to evaluate the performance of these plasma cfDNA fungal PCR assays in pediatric oncology and hematopoietic stem cell transplant (HSCT) patients with clinical concern for IFD. Methods We initiated an observational study of inpatient oncology and HSCT patients who had plasma mold panel (+/- Candida panel) cfDNA fungal PCR obtained as part of an IFD evaluation at our freestanding quaternary care children’s hospital. The primary outcome was IFD clinical diagnosis (proven, probable, possible, unlikely) as per EORTC/MSG† definitions within one month of the cfDNA fungal PCR, which was assigned independently by two physicians, with a third physician utilized in cases of discrepancy. Patient demographics, hospital course, imaging results, and clinical laboratory data were abstracted and maintained in an encrypted REDCap© database. Results In a preliminary analysis of October 2021-March 2022 data, there were 21 IFD evaluations for 18 patients (Table 2). Most oncology evaluations were for prolonged febrile neutropenia, while many HSCT were non-neutropenic, but on enhanced immunosuppression with new clinical concerns (e.g., respiratory symptoms, persistent fever). Plasma cfDNA detected a mold or yeast consistent with the clinical presentation in 100% of the five proven/probable cases (Figures 1 &amp; 2). All 14 possible IFD cases had a negative cfDNA fungal PCR. Proven cases are designated with blue icon, while the probable case is orange. All five cases were in oncology patients who did not have history of hematopoietic stem cell transplant. Cell free DNA (cfDNA) fungal polymerase chain reaction (PCR) results include the organism identified, followed by the cycle threshold (Ct) in parenthesis. Maximum Ct for the assay is 45. Abbreviations: AG: Aspergillus galactomannan index (Ref &amp;lt;0.5); ALL: acute lymphoblastic leukemia. BAL: bronchoalveolar lavage; 1,3-BDG: 1,3-beta-D-glucan (Ref &amp;lt;60 pg/ml); cfDNA: cell free deoxyribonucleic acid; CNS: central nervous system; EORTC/MSG: European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group &amp; the National Institute of Allergy &amp; Infectious Diseases Mycoses Study Group; L-AmB: liposomal amphotericin B; SARS-CoV-2: severe acute respiratory syndrome associated with coronavirus 2. Conclusion Upfront plasma cfDNA fungal PCR successfully detected a relevant mold or yeast with short turnaround time in 100% of cases that were later classified as proven/probable. This appears promising for early, noninvasive diagnosis that enables prompt optimization of antifungal and surgical management, particularly for mucormycosis cases. Additional data may permit consideration of Mucorales agents PCR as a new EORTC/MSG mycologic criteria. Disclosures Lauren E. Kushner, MD, Roman: Client of my husband's business..

Clinical Impact of Noninvasive Plasma Microbial Cell-Free Deoxyribonucleic Acid Sequencing for the Diagnosis and Management of Pneumocystis jirovecii Pneumonia: A Single-Center Retrospective Study.

We present 23 cases of Pneumocystis jirovecii pneumonia (PCP) diagnosed with commercially available noninvasive plasma microbial cell-free deoxyribonucleic acid (mcfDNA) assay. Our findings suggest that plasma mcfDNA testing resulted in positive clinical impact for the diagnosis and treatment of PCP and coinfections in 82.6% of cases.