Measurement Biases Distort Cell-Free DNA Fragmentation Profiles and Define the Sensitivity of Metagenomic Cell-Free DNA Sequencing Assays.

BACKGROUND: Metagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing. METHODS: We characterized the fragment length biases inherent to select DNA isolation and library preparation procedures and developed a model to correct for these biases. We analyzed 305 cfDNA sequencing data sets, including publicly available data sets and 124 newly generated data sets, to evaluate the dependence of the sensitivity of metagenomic cfDNA sequencing on pre-analytical variables. RESULTS: Length bias correction of fragment length distributions measured from different experimental procedures revealed the ultrashort (<100 bp) nature of microbial-, mitochondrial-, and host-specific urinary cfDNA. The sensitivity of metagenomic sequencing assays to detect the clinically reported microorganism differed by more than 5-fold depending on the combination of DNA isolation and library preparation used. CONCLUSIONS: Substantial gains in the sensitivity of microbial and other short fragment recovery can be achieved by easy-to-implement changes in the sample preparation protocol, which highlights the need for standardization in the liquid biopsy field.

Using MRSA Screening Tests To Predict Methicillin Resistance in Staphylococcus aureus Bacteremia.

Bloodstream infections with Staphylococcus aureus are clinically significant and are often treated with empirical methicillin resistance (MRSA, methicillin-resistant S. aureus) coverage. However, vancomycin has associated harms. We hypothesized that MRSA screening correlated with resistance in S. aureus bacteremia and could help determine the requirement for empirical vancomycin therapy. We reviewed consecutive S. aureus bacteremias over a 5-year period at two tertiary care hospitals. MRSA colonization was evaluated in three ways: as tested within 30 days of bacteremia (30-day criterion), as tested within 30 days but accounting for any prior positive results (ever-positive criterion), or as tested in known-positive patients, with patients with unknown MRSA status being labeled negative (known-positive criterion). There were 409 S. aureus bacteremias: 302 (73.8%) methicillin-susceptible S. aureus (MSSA) and 107 (26.2%) MRSA bacteremias. In the 167 patients with MSSA bacteremias, 7.2% had a positive MRSA test within 30 days. Of 107 patients with MRSA bacteremia, 68 were tested within 30 days (54 positive; 79.8%), and another 21 (19.6%) were previously positive. The 30-day criterion provided negative predictive values (NPV) exceeding 90% and 95% if the prevalence of MRSA in S. aureus bacteremia was less than 33.4% and 19.2%, respectively. The same NPVs were predicted at MRSA proportions below 39.7% and 23.8%, respectively, for the ever-positive criterion and 34.4% and 19.9%, respectively, for the known-positive criterion. In MRSA-colonized patients, positive predictive values exceeded 50% at low prevalence. MRSA screening could help avoid empirical vancomycin therapy and its complications in stable patients and settings with low-to-moderate proportions of MRSA bacteremia.

Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C.

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here, we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with COVID-19 or MIS-C across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multiorgan involvement in MIS-C encompassing diverse cell types, including endothelial and neuronal cells, and an enrichment of pyroptosis-related genes. Whole-blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C but also MIS-C-specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole-blood RNA in paired samples yields different but complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs future development of new disease biomarkers.

UDP-glucose dehydrogenase (UGDH) activity is suppressed by peroxide and promoted by PDGF in fibroblast-like synoviocytes: Evidence of a redox control mechanism.

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.

Peritoneal Effluent Cell-Free DNA Sequencing in Peritoneal Dialysis Patients With and Without Peritonitis.

RATIONALE & OBJECTIVE: Conventional culture can be insensitive for the detection of rare infections and for the detection of common infections in the setting of recent antibiotic usage. Patients receiving peritoneal dialysis (PD) with suspected peritonitis have a significant proportion of negative conventional cultures. This study examines the utility of metagenomic sequencing of peritoneal effluent cell-free DNA (cfDNA) for evaluating the peritoneal effluent in PD patients with and without peritonitis. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: We prospectively characterized cfDNA in 68 peritoneal effluent samples obtained from 33 patients receiving PD at a single center from September 2016 to July 2018. OUTCOMES: Peritoneal effluent, microbial, and human cfDNA characteristics were evaluated in culture-confirmed peritonitis and culture-negative peritonitis. ANALYTICAL APPROACH: Descriptive statistics were analyzed and microbial cfDNA was detected in culture-confirmed peritonitis and culture-negative peritonitis. RESULTS: Metagenomic sequencing of cfDNA was able to detect and identify bacterial, viral, and eukaryotic pathogens in the peritoneal effluent from PD patients with culture-confirmed peritonitis, as well as patients with recent antibiotic usage and in cases of culture-negative peritonitis. LIMITATIONS: Parallel cultures were not obtained in all the peritoneal effluent specimens. CONCLUSIONS: Metagenomic cfDNA sequencing of the peritoneal effluent can identify pathogens in PD patients with peritonitis, including culture-negative peritonitis.

Lightning does strike twice: leveraging phased variants to enhance minimal residual disease detection.

Liquid biopsy detection of residual cancer after therapy offers to transform oncology care. Nonetheless, in the residual cancer context, signals are sparse and are hindered by technical sequencing noise. Kurtz et al.1 introduce phased variant enrichment and detection sequencing (phasED-seq) to increase the circulating tumor DNA signal-to-noise ratio and detect minimal residual disease with unprecedented sensitivity.

Beta-Lactam/Beta-Lactamase Inhibitor Therapy for Potential AmpC-Producing Organisms: A Systematic Review and Meta-Analysis.

The optimal treatment for potential AmpC-producing Enterobacteriaceae, including Serratia, Providencia, Citrobacter, Enterobacter, and Morganella species, remains unknown. An updated systematic review and meta-analysis of studies comparing beta-lactam/beta-lactamase inhibitors with carbapenems in the treatment of bloodstream infections with these pathogens found no significant difference in 30-day mortality (OR, 1.13; 95% CI, 0.58 - 2.20).

Lysosomal rupture induced by structurally distinct chitosans either promotes a type 1 IFN response or activates the inflammasome in macrophages.

Chitosan is a family of glucosamine and N-acetyl glucosamine polysaccharides with poorly understood immune modulating properties. Here, functional U937 macrophage responses were analyzed in response to a novel library of twenty chitosans with controlled degree of deacetylation (DDA, 60-98%), molecular weight (1 to >100 kDa), and acetylation pattern (block vs. random). Specific chitosan preparations (10 or 190 kDa 80% block DDA and 3, 5, or 10 kDa 98% DDA) either induced macrophages to release CXCL10 and IL-1ra at 5-50 µg/mL, or activated the inflammasome to release IL-1ß and PGE2 at 50-150 µg/mL. Chitosan induction of these factors required lysosomal acidification. CXCL10 production was preceded by lysosomal rupture as shown by time-dependent co-localization of galectin-3 and chitosan and slowed autophagy flux, and specifically depended on IFN-ß paracrine activity and STAT-2 activation that could be suppressed by PGE2. Chitosan induced a type I IFN paracrine response or inflammasome response depending on the extent of lysosomal rupture and cytosolic foreign body invasion. This study identifies the structural motifs that lead to chitosan-driven cytokine responses in macrophages and indicates that lysosomal rupture is a key mechanism that determines the endogenous release of either IL-1ra or IL-1ß.

Back to the Future: Penicillin-Susceptible Staphylococcus aureus.

BACKGROUND: Widespread penicillin usage rapidly resulted in the emergence of penicillin resistance in Staphylococcus aureus. However, new data suggest that penicillin susceptibility may be in a period of renaissance. The objective of our study was to quantify penicillin resistance in methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. METHODS: We retrospectively reviewed all adult MSSA bacteremia from April 2010 to April 2015 at the McGill University Health Centre (Montreal, QC, Canada). Susceptibility to penicillin, erythromycin, clindamycin, and trimethoprim-sulfamethoxazole (TMP-SMX) was determined in accordance with the Clinical & Laboratory Standards Institute guidelines. RESULTS: There were 324 unique episodes of MSSA bacteremia. Ninety (28%) isolates were susceptible to penicillin, 229 (71%) to erythromycin, 239 (74%) to clindamycin, and 317 (98%) to TMP-SMX. Isolates that were penicillin resistant were more likely to also be resistant to other antibiotics, but a statistically significant association was apparent only for erythromycin resistance (76/234, 32.2% vs 19/90, 21.1%, P = .04). The median age of patients was 67.5 years (interquartile range 52-78) and overall in-hospital 30-day mortality was 16.3% (53 deaths). After adjustment for patient age, there was no association between penicillin resistance and either intensive care unit admission or death. CONCLUSION: More than one-quarter of patients with MSSA bacteremia potentially could be treated with parenteral penicillin, which may offer pharmacokinetic advantages over other beta-lactam drugs and potentially improved outcomes.