Fragment Ends of Circulating Microbial DNA as Signatures for Pathogen Detection in Sepsis.

BACKGROUND: Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. METHODS: We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. RESULTS: The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. CONCLUSIONS: The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.

Jagged Ends on Multinucleosomal Cell-Free DNA Serve as a Biomarker for Nuclease Activity and Systemic Lupus Erythematosus.

BACKGROUND: Jagged ends of plasma DNA are a recently recognized class of fragmentomic markers for cell-free DNA, reflecting the activity of nucleases. A number of recent studies have also highlighted the importance of jagged ends in the context of pregnancy and oncology. However, knowledge regarding the generation of jagged ends is incomplete. METHODS: Jaggedness of plasma DNA was analyzed based on Jag-seq, which utilized the differential methylation signals introduced by the DNA end-repair process. We investigated the jagged ends in plasma DNA using mouse models by deleting the deoxyribonuclease 1 (Dnase1), DNA fragmentation factor subunit beta (Dffb), or deoxyribonuclease 1 like 3 (Dnase1l3) gene. RESULTS: Aberrations in the profile of plasma DNA jagged ends correlated with the type of nuclease that had been genetically deleted, depending on nucleosomal structures. The deletion of Dnase1l3 led to a significant reduction of jaggedness for those plasma DNA molecules involving more than 1 nucleosome (e.g., size ranges 240-290 bp, 330-380 bp, and 420-470 bp). However, less significant effects of Dnase1 and Dffb deletions were observed regarding different sizes of DNA fragments. Interestingly, the aberration in plasma DNA jagged ends related to multinucleosomes was observed in human subjects with familial systemic lupus erythematosus with Dnase1l3 deficiency and human subjects with sporadic systemic lupus erythematosus. CONCLUSIONS: Detailed understanding of the relationship between nuclease and plasma DNA jaggedness has opened up avenues for biomarker development.

Effects of early corticosteroid treatment on plasma SARS-associated Coronavirus RNA concentrations in adult patients.

BACKGROUND: The effect of corticosteroid treatment on the viral load of Severe Acute Respiratory Syndrome (SARS) patients is unknown. OBJECTIVE: To compare the plasma SARS-CoV RNA concentrations in ribavirin-treated patients who received early hydrocortisone therapy with those who received placebo. STUDY DESIGN: Serial plasma SARS-CoV RNA concentrations measured in the setting of a prospective, randomized double-blinded, placebo-controlled trial designed to assess the efficacy of "early" (<7 days of illness) hydrocortisone use in previously healthy SARS patients were analyzed. SARS-CoV RNA was quantified using a one-step real-time RT-PCR assay targeting the nucleocapsid gene. RESULTS: Among 16 non-ICU cases, SARS-CoV RNA was detected in plasma since day 3-4 after fever onset; viral concentration peaked in the first week, which then rapidly declined in the second week of illness. On days 8, 12, 16, and 20, the cumulative proportion of patients with undetectable virus in plasma was 31%, 69%, 92%, and 100%, respectively. Plasma SARS-CoV RNA concentrations in the second and third week of illness were significantly higher in patients who received initial hydrocortisone treatment (n = 9), as compared to those who received placebo (n = 7)(AUC; Mann-Whitney, P = 0.023). The median time for SARS-CoV to become undetectable in plasma was 12 days (11-20 days) versus 8 days (8-15 days), respectively. CONCLUSION: Our findings suggested "early" corticosteroid treatment was associated with a higher subsequent plasma viral load.